Prefusion coronavirus spike proteins and their use

ABSTRACT

Coronavirus S ectodomain trimers stabilized in a prefusion conformation, nucleic acid molecules and vectors encoding these proteins, and methods of their use and production are disclosed. In several embodiments, the coronavirus S ectodomain trimers and/or nucleic acid molecules can be used to generate an immune response to coronavirus in a subject. In additional embodiments, the therapeutically effective amount of the coronavirus S ectodomain trimers and/or nucleic acid molecules can be administered to a subject in a method of treating or preventing coronavirus infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/412,703, filed Oct. 25, 2016, which is herein incorporated byreference in its entirety.

FIELD OF THE DISCLOSURE

This disclosure relates to recombinant coronavirus spike (S) proteins,such as Middle East respiratory syndrome coronavirus (MERS-CoV) andsevere acute respiratory syndrome coronavirus (SARS-CoV) S proteins,that are stabilized in a prefusion conformation by one or more aminoacid substitutions, and their use as immunogens.

BACKGROUND

Coronaviruses are enveloped, positive-sense single-stranded RNA viruses.They have the largest genomes (26-32 kb) among known RNA viruses, andare phylogenetically divided into four genera (α, β, γ, δ), withbetacoronaviruses further subdivided into four lineages (A, B, C, D).Coronaviruses infect a wide range of avian and mammalian species,including humans. Of the six known human coronaviruses, four of them(HCoV-OC43, HCoV-229E, HCoV-HKU1 and HCoV-NL63) circulate annually inhumans and generally cause mild respiratory diseases, although severitycan be greater in infants, elderly, and the immunocompromised. Incontrast, the Middle East respiratory syndrome coronavirus (MERS-CoV)and the severe acute respiratory syndrome coronavirus (SARS-CoV),belonging to betacoronavirus lineages C and B, respectively, are highlypathogenic. Both viruses emerged into the human population from animalreservoirs within the last 15 years and caused outbreaks with highcase-fatality rates.

MERS-CoV was isolated in 2012 from a patient in Saudi Arabia and isstill circulating across the Arabian Peninsula. Primary transmission,most likely from camels, is now considered to be the most common routeof transmission, and camels are thought to be a secondary orintermediate reservoir for MERS-CoV, with bats serving as the primaryreservoir. Human-to-human transmission, especially as a result of closecontact between patients and hospital workers within health caresettings, is another important route of transmission, and wasresponsible for an outbreak of MERS-CoV in South Korea. The highpathogenicity and airborne transmissibility of SARS-CoV and MERS-CoVhave raised concern about the potential for another coronaviruspandemic. The high case-fatality rate, vaguely defined epidemiology, andabsence of prophylactic or therapeutic measures against coronaviruseshave created an urgent need for an effective vaccine and relatedtherapeutic agents.

SUMMARY

Disclosed herein are recombinant coronavirus S ectodomain trimerscomprising protomers comprising one or more proline substitution(s) thatstabilize the S protein trimer in the prefusion conformation. One classof mutation, comprising one or more (such as two) proline substitutionsat or near the boundary between a Heptad Repeat 1 (HR1) and a centralhelix of the protomers of the coronavirus S ectodomain trimer was foundto be surprisingly effective for stabilization of coronavirus S proteintrimers in the prefusion conformation. Embodiments of suchprefusion-stabilized coronavirus S ectodomain trimers are demonstratedto produce a superior immune response in an animal model compared tocorresponding coronavirus S ectodomain trimers that are not stabilizedin the prefusion conformation.

In some embodiments, an immunogen is provided that comprises arecombinant alphacoronavirus or betacoronavirus S ectodomain trimercomprising protomers comprising one or two proline substitutions at ornear a junction between a heptad repeat 1 (HR1) and a central helix thatstabilize the S ectodomain trimer in a prefusion conformation. The oneor two proline substitutions can comprise two consecutive prolinesubstitutions (a “double proline substitution”). In some embodiments,the recombinant alphacoronavirus or betacoronavirus S ectodomain trimercomprises S ectodomains from a NL63-CoV, 229E-CoV, OC43-CoV, SARS-CoV,MERS-CoV, HKU1-CoV, WIV1-CoV, mouse hepatitis virus (MHV), or HKU9-CoV,that comprise the one or two proline substitutions.

In some embodiments, the recombinant alphacoronavirus or betacoronavirusS ectodomain trimer comprises: a recombinant HKU1-CoV S ectodomaintrimer, and the double proline substitution is between residues 1050 to1070 of the protomers in the trimer (for example, N1067P and L1068Psubstitutions); a recombinant SARS-CoV S ectodomain trimer, and thedouble proline substitution is between residues 951 to 971 of theprotomers in the trimer (for example, K968P and V969P substitutions); arecombinant MERS-CoV S ectodomain trimer, and the double prolinesubstitution is between residues 1050 to 1069 of the protomers in thetrimer (for example, V1060P and L1061P substitutions); a recombinantOC43-CoV S ectodomain trimer, and the double proline substitution isbetween residues 1062 to 1082 of the protomers in the trimer (forexample, A1079P and L1080P substitutions); a recombinant HKU9-CoV Sectodomain trimer, and the double proline substitution is betweenresidues 966 to 986 of the protomers in the trimer (for example, G1018Pand L1019P substitutions); a recombinant NL63-CoV S ectodomain trimer,and the double proline substitution is between residues 1035 to 1055 ofthe protomers in the trimer (for example, S1052P and I1053Psubstitutions); a recombinant 229E-CoV S ectodomain trimer, and thedouble proline substitution is between residues 852 to 872 of theprotomers in the trimer (for example, I869P and I870P substitutions); arecombinant WIV1-CoV S ectodomain trimer, and the double prolinesubstitution is between residues 952 to 972 of the protomers in thetrimer (for example, K969P and V970P substitutions); or a recombinantMHV S ectodomain trimer, and the double proline substitution is betweenresidues 852 to 872 of the protomers in the trimer (for example, I869Pand I870P substitutions).

In some embodiments, the protomers of the recombinant alphacoronavirusor betacoronavirus S ectodomain trimer further comprise one or moreadditional amino acid substitutions or deletions, such as amino acidsubstitutions that stabilize the recombinant alphacoronavirus orbetacoronavirus S ectodomain trimer in the prefusion conformation, oramino acid substitutions to inhibit or prevent protease cleavage at aS1/S2 protease cleavage site and/or a S2′ protease cleave site of the Sectodomain.

In some embodiments, the protomers of the recombinant alphacoronavirusor betacoronavirus S ectodomain trimer can be linked to a trimerizationdomain (such as T4 Fibritin trimerization domain). In additionalembodiments, the protomers of the recombinant alphacoronavirus orbetacoronavirus S ectodomain trimer can be linked to a transmembranedomain.

In additional embodiments, the recombinant coronavirus S ectodomaintrimer can be included on a protein nanoparticle, such as a ferritinprotein nanoparticle. Nucleic acid molecules encoding a protomer of thedisclosed recombinant coronavirus S ectodomain trimers are alsoprovided, as are vectors including the nucleic acid molecules, andmethods of producing the disclosed coronavirus S ectodomain trimers.

Immunogenic compositions including the recombinant coronavirus Sectodomain trimer that are suitable for administration to a subject arealso provided, and may also be contained in a unit dosage form. Thecompositions can further include an adjuvant. The recombinantcoronavirus S ectodomain trimers may also be conjugated to a carrier tofacilitate presentation to the immune system.

Methods of inducing an immune response in a subject are disclosed, asare methods of treating, inhibiting or preventing a coronavirusinfection in a subject, by administering to the subject an effectiveamount of a disclosed recombinant coronavirus S ectodomain trimer,nucleic acid molecule, or vector.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C illustrate the structure of the HKU1-CoV prefusion spikeectodomain. (1A) A single protomer of the trimeric S protein is shown incartoon representation colored as a rainbow from the N to C terminus(blue to red) with the reconstructed EM density of remaining protomersshown in white and grey. (1B) The S1 subunit is composed of theN-terminal domain (NTD) and C-terminal domain (CTD) as well as twosub-domains (SD-1 and SD-2). The S2 subunit contains the coronavirusfusion machinery and is primarily α-helical. (1C) Domain architecture ofthe HKU1-CoV S protein colored as in (1A).

FIGS. 2A-2D illustrate the architecture of the HKU1-CoV S1 subunit. (2A)EM density corresponding to each S1 protomer is shown. The putativeglycan-binding and protein-receptor-binding sites are indicated withdashed shapes on the NTD and CTD, respectively. (2B) The HKU1-CoV S1 CTDforms quaternary interactions with an adjacent CTD using a surfacesimilar to that used by SARS-CoV CTD to bind its receptor, ACE2. (2C)SD-1 is composed of amino acid residues before and after the S1 CTD.(2D) SD-2 is composed of S1 sequence C-terminal to the CTD, a shortpeptide following the NTD, and the N-terminal strand of S2, whichfollows the S1/S2 furin-cleavage site.

FIGS. 3A-3C illustrate the HKU1-CoV S2 subunit fusion machinery. (3A)The HKU1-CoV S2 subunit is colored like a rainbow from the N-terminalβ-strand (blue), which participates in S1 sub-domain 2, to the Cterminus (red) before HR2. (3B) The HKU1-CoV S2 structure contains thefusion peptide (FP) and a HR1. Protease-recognition sites are indicatedwithin disordered regions of the protein (dashed lines). (3C) Acomparison of coronavirus S2 HR1 in the pre- and post-fusionconformations. Five HR1 α-helices are labelled and colored like arainbow from blue to red, N to C terminus, respectively. The structuresare oriented to position similar portions of the central helix (red).

FIGS. 4A-4C illustrate stabilization of MERS-CoV S protein in aprefusion conformation by V1060P (“Top 3”) and L1061P (“Top 4”) aminoacid substitutions. (4A) Location of various stabilization designconceptions. V1060P and L1061P (red circle) are located at the top of S2HR1 and the S2 central helix. MERS-CoV S ectodomains with V1060P andL1061P mutations were expressed individually and in combination andpurified. Protein expression levels and purity were determined by (4B)gel electrophoresis and (4C) size-exclusion chromatography.

FIGS. 5A-5B are a set of graphs showing results from neutralizationassays using sera from mice immunized with the MERS-CoV S prefusionstabilized (2P) ectodomain trimer. Mice (N=5/group) were immunized with0.1 μg of MERS-CoV wild-type S ectodomain trimer or MERS-CoVprefusion-stabilized S ectodomain trimer intramuscularly with SigmaAdjuvant System at weeks 0 and 3. Control mice were given PBS. Two weeksfollowing the last immunization, serum was collected and tested forneutralizing antibodies against various MERS pseudovirus strains:England1, Florida USA2, Bisha1, Korea002, JordanN3, Buraidah1, andIndiana USA1. (FIG. 5A) Reciprocal serum IC₉₀ neutralizing activityagainst autologous MERS England1 pseudotyped lentivirus reporter plottedagainst vaccine dose. (FIG. 5B) Reciprocal serum IC₉₀ neutralizingactivity against multiple homologous MERS-CoV pseudoviruses of sera frommice immunized with 0.1 μg of purified MERS-CoV S ectodomain trimer. Forboth panels, the geometric mean IC₉₀ titer (GMT) of each group isrepresented by (FIG. 5A) symbols or (FIG. 5B) bars. Error bars representgeometric SDs. P values denoted as *P<0.05 and **P<0.01. The limit ofdetection for the assay is represented by dotted lines; for sera belowthe limit of detection a reciprocal IC₉₀ titer of 10 was assigned.

FIGS. 6A and 6B shows results from the dissection of binding andneutralizing antibodies elicited by MERS S-2P. Serum from mice immunizedwith (A) MERS 51, (B) MERS S WT ectodomain trimer, and MERS S-2Pectodomain trimer were depleted of MERS RBD, MERS 51, and MERS S-2Pectodomain trimer specific antibodies by magnetic bead depletion. Theresulting depleted serum was then tested for (FIG. 6A) MERS S-2Pectodomain trimer specific antibodies by ELISA or (FIG. 6B) neutralizingantibodies against MERS England1 pseudovirus. For the binding assays.endpoint ELISA titers were determined, and % binding retained wascalculated as a measure of endpoint titers for each serum depleted withMERS protein compared to binding after depletion with a nonspecificprotein. For the neutralization assays, IC50 titers were determined, and% neutralization retained was calculated as a measure of neutralizationeach serum depleted with MERS protein compared to binding afterdepletion with a nonspecific protein. Bars represent the mean of eachgroup; error bars represent SD.

FIG. 7 is a set of graphs showing that MERS-CoV S-2P immunizationprotects against lethal MERS challenge in mice. C57BL/6J mice weregenetically engineered using CRISPR-Cas9 genomic editing to encode humanDPP4 mutations (288L and 330R; “288/330^(+/+)”) as previously described(see, Cockrell et al., “A mouse model for MERS coronavirus-induced acuterespiratory distress syndrome.” Nature Microbiology. 2:16226, 2016,which is incorporated by reference herein). 288/330^(+/+) mice werevaccinated with 0.1 μg MERS-CoV S-2P or PBS, with Sigma Adjuvant Systemat weeks 0 and 3. Four weeks following final vaccination, mice werechallenged with a lethal dose of mouse-adapted MERS virus and monitoredfor survival and weight loss.

FIG. 8 illustrates the structural domains of the HKU1-CoV, SARS-CoV, andMERS-CoV S proteins, as well as positioning of double prolinesubstitutions to stabilize these proteins in the prefusion conformation.

FIGS. 9A-9C show a sequence alignment of the S2 subunit of the HKU1-CoV(SEQ ID NO: 8), SARS-CoV (SEQ ID NO: 6), MERS-CoV (SEQ ID NO: 1),HKU9-CoV (SEQ ID NO: 12), NL63-CoV (SEQ ID NO: 18), and 229E-CoV (SEQ IDNO: 20) S proteins, showing relevant sequence homology.

FIG. 10 shows a Coomassie-stained polyacrylamide gel illustrating thatintroduction of proline substitutions in the SARS-CoV (K968P and V969Psubstitutions, SARS-S-2P) and HKU1-CoV (N1067P and L1068P substitutions,HKU1-S-2P) S ectodomains at the locations corresponding to the MERS-CoVS V1060P and L1061P substitutions boosts the expression of the SARS-CoVand HKU1-CoV S ectodomains.

FIG. 11 shows a Coomassie-stained polyacrylamide gel illustrating thatthe SARS-CoV S ectodomain with K968P and V969P substitutions (SARS-S-2P)has higher thermal stability than corresponding SARS-CoV S ectodomainhaving native sequence (SARS-S-WT).

FIG. 12 shows a set of graphs illustrating gel chromatography results ofpurified SARS-CoV, MERS-CoV, and HKU1-CoV S ectodomains having native(S-WT) sequence or double proline substitutions noted above (S-2P).

FIGS. 13A-13C show images of negative-stain electron microscopy ofpurified ectodomain trimers of MERS-CoV S 2P (V1060P and L1061P, SEQ IDNO: 28), SARS-CoV S 2P (K968P and V969P, SEQ ID NO: 30), HKU1-CoV S 2P(N1067P and L1068P, SEQ ID NO: 31), OC43-CoV S 2P (A1079P and L1080P,SEQ ID NO: 33), WIV1-CoV S 2P (K969P and V970P, SEQ ID NO: 34), PEDV-CoVS 2P (I1076P and L1077P, SEQ ID NO: 40), 229E S-2P (I869P and 1870P, SEQID NO: 37), and SDCV 2-2P. Each of these ectodomain trimers was purifiedas a single peak and formed trimers in the typical prefusionconformation.

FIGS. 14A-14G show low-resolution negative-stain reconstructions of Strimer constructs from (14A) HKU1-CoV S 2P ectodomain trimer, (14B)MERS-CoV S 2P ectodomain trimer, (14C) SARS-CoV S 2P ectodomain trimer,(14D) OC43 S-2P ectodomain trimer, (14E) WIV1-CoV S 2P ectodomaintrimer, (14F) PEDV-CoV S 2P ectodomain trimer, and (14G) 229E-CoV S 2Pectodomain trimer that were obtained from the negative-stain electronmicroscopy data shown in FIG. 13. The particles all formed homogeneoustrimeric spike protein structures.

FIG. 15 is a graph showing results of immunogenicity assays of HKU1-CoVS 2P ectodomain trimer and SARS S-2P ectodomain trimer in mice.Reciprocal serum IC₉₀ neutralizing activity against autologouspseudotyped lentivirus reporter (SARS Urbani for the SARS immunization)plotted against vaccine dose. The geometric mean IC₉₀ titer (GMT) ofeach group is represented by symbols. Error bars represent geometricSDs. The limit of detection for the assay is represented by dottedlines; for sera below the limit of detection a reciprocal IC₉₀ titer of10 was assigned.

FIG. 16 shows results from immunogenicity assays in mice using theOC43-CoV S-2P and WIV1-CoV S-2P ectodomain trimer immunogens. BALB/cmice were vaccinated with 1 μg of OC43 S-2P ectodomain trimer orWIV1-CoV S-2P ectodomain trimer, with Sigma Adjuvant System at weeks 0and 3. Two weeks following final vaccination, mice were bled forantibody analysis. Binding antibody titers to OC43 S-2P ectodomaintrimer or WIV1-CoV S-2P ectodomain trimer were measured by ELISA. Thegeometric mean titer (GMT) and geometric SDs of each group arerepresented. The dotted line represents the assay limit of detection. **denotes p-value <0.01, determined by Mann-Whitney t-test.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile in the form of the file named “Sequence.txt” (˜404 kb), which wascreated on Oct. 24, 2017, and which is incorporated by reference herein.

DETAILED DESCRIPTION

Past efforts to develop coronavirus vaccines have used whole-inactivatedvirus, live-attenuated virus, recombinant protein subunit, or geneticapproaches (Graham et al., Nature reviews. Microbiology 11, 836, 2013).This disclosure provides CoV Spike glycoprotein (S) ectodomain trimersthat are stabilized in the prefusion conformation and which are shown toelicit a neutralizing immune response in animal models.

I. Summary of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes X, published by Jones & BartlettPublishers, 2009; and Meyers et al. (eds.), The Encyclopedia of CellBiology and Molecular Medicine, published by Wiley-VCH in 16 volumes,2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.” As used herein, the term “comprises” means “includes.” It isfurther to be understood that any and all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescriptive purposes, unless otherwise indicated. Although many methodsand materials similar or equivalent to those described herein can beused, particular suitable methods and materials are described herein. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting. To facilitatereview of the various embodiments, the following explanations of termsare provided:

Adjuvant: A vehicle used to enhance antigenicity. In some embodiments,an adjuvant can include a suspension of minerals (alum, aluminumhydroxide, or phosphate) on which antigen is adsorbed; or water-in-oilemulsion, for example, in which antigen solution is emulsified inmineral oil (Freund incomplete adjuvant), sometimes with the inclusionof killed mycobacteria (Freund's complete adjuvant) to further enhanceantigenicity (inhibits degradation of antigen and/or causes influx ofmacrophages). In some embodiments, the adjuvant used in a disclosedimmunogenic composition is a combination of lecithin and carbomerhomopolymer (such as the ADJUPLEX™ adjuvant available from AdvancedBioAdjuvants, LLC, see also Wegmann, Clin Vaccine Immunol, 22(9):1004-1012, 2015). Additional adjuvants for use in the disclosedimmunogenic compositions include the QS21 purified plant extract, MatrixM, ASO1, MF59, and ALFQ adjuvants. Immunostimulatory oligonucleotides(such as those including a CpG motif) can also be used as adjuvants.Adjuvants include biological molecules (a “biological adjuvant”), suchas costimulatory molecules. Exemplary adjuvants include IL-2, RANTES,GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL andtoll-like receptor (TLR) agonists, such as TLR-9 agonists. Additionaldescription of adjuvants can be found, for example, in Singh (ed.)Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007).Adjuvants can be used in combination with the disclosed immunogens.

Administration: The introduction of an agent, such as a disclosedimmunogen, into a subject by a chosen route. Administration can be localor systemic. For example, if the chosen route is intranasal, the agent(such as an immunogen comprising a recombinant coronavirus S ectodomaintrimer stabilized in a prefusion conformation) is administered byintroducing the composition into the nasal passages of the subject.Exemplary routes of administration include, but are not limited to,oral, injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal, and intravenous), sublingual, rectal, transdermal (forexample, topical), intranasal, vaginal, and inhalation routes.

Amino acid substitution: The replacement of one amino acid in apolypeptide with a different amino acid.

Antibody: An immunoglobulin, antigen-binding fragment, or derivativethereof, that specifically binds and recognizes an analyte (antigen)such as a coronavirus S protein, an antigenic fragment thereof, or adimer or multimer of the antigen. The term “antibody” is used herein inthe broadest sense and encompasses various antibody structures,including but not limited to monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments, so long as they exhibit the desired antigen-bindingactivity. Non-limiting examples of antibodies include, for example,intact immunoglobulins and variants and fragments thereof that retainbinding affinity for the antigen. Examples of antibody fragments includebut are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies;linear antibodies; single-chain antibody molecules (e.g. scFv); andmultispecific antibodies formed from antibody fragments. Antibodyfragments include antigen binding fragments either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed),Antibody Engineering, Vols. 1-2, 2^(nd) Ed., Springer Press, 2010).

Carrier: An immunogenic molecule to which an antigen can be linked. Whenlinked to a carrier, the antigen may become more immunogenic. Carriersare chosen to increase the immunogenicity of the antigen and/or toelicit antibodies against the carrier which are diagnostically,analytically, and/or therapeutically beneficial. Useful carriers includepolymeric carriers, which can be natural (for example, proteins frombacteria or viruses), semi-synthetic or synthetic materials containingone or more functional groups to which a reactant moiety can beattached.

Cavity-filling amino acid substitution: An amino acid substitution thatfills a cavity within the protein core of a protein, such as acoronavirus S protein ectodomain. Cavities are essentially voids withina folded protein where amino acids or amino acid side chains are notpresent. In several embodiments, a cavity-filling amino acidsubstitution is introduced to fill a cavity present in the prefusionconformation of a coronavirus S ectodomain core that collapses (e.g.,has reduced volume) after transition to the postfusion conformation.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease a function ofa protein, such as the ability of the protein to induce an immuneresponse when administered to a subject. The term conservative variationalso includes the use of a substituted amino acid in place of anunsubstituted parent amino acid. Furthermore, deletions or additionswhich alter, add or delete a single amino acid or a small percentage ofamino acids (for instance less than 5%, in some embodiments less than1%) in an encoded sequence are conservative variations where thealterations result in the substitution of an amino acid with achemically similar amino acid.

The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity orfunction of the recombinant coronavirus S ectodomain trimer, such as theability to induce an immune response when administered to a subject. Forinstance, if an amino acid residue is essential for a function of theprotein, even an otherwise conservative substitution may disrupt thatactivity. Thus, a conservative substitution does not alter the basicfunction of a protein of interest.

Control: A reference standard. In some embodiments, the control is anegative control sample obtained from a healthy patient. In otherembodiments, the control is a positive control sample obtained from apatient diagnosed with a coronavirus infection, such as MERS-CoV orSARS-CoV. In still other embodiments, the control is a historicalcontrol or standard reference value or range of values (such as apreviously tested control sample, such as a group of patients infectedwith a coronavirus with known prognosis or outcome, or group of samplesthat represent baseline or normal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%.

Coronavirus: A family of positive-sense, single-stranded RNA virusesthat are known to cause severe respiratory illness. Viruses currentlyknown to infect human from the coronavirus family are from thealphacoronavirus and betacoronavirus genera. Additionally, it isbelieved that the gammacoronavirus and deltacoronavirus genera mayinfect humans in the future.

Non-limiting examples of betacoronaviruses include Middle Eastrespiratory syndrome coronavirus (MERS-CoV), Severe Acute RespiratorySyndrome coronavirus (SARS-CoV), Human coronavirus HKU1 (HKU1-CoV),Human coronavirus OC43 (OC43-CoV), Murine Hepatitis Virus (MHV-CoV), BatSARS-like coronavirus WIV1 (WIV1-CoV), and Human coronavirus HKU9(HKU9-CoV). Non-limiting examples of alphacoronaviruses include humancoronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), porcineepidemic diarrhea virus (PEDV), and Transmissible gastroenteritiscoronavirus (TGEV). A non-limiting example of a deltacoronaviruses isthe Swine Delta Coronavirus (SDCV). Exemplary sequences of theectodomains of S proteins from these viruses are provided herein.

The viral genome is capped, polyadenylated, and covered withnucleocapsid proteins. The coronavirus virion includes a viral envelopecontaining type I fusion glycoproteins referred to as the spike (S)protein. Most coronaviruses have a common genome organization with thereplicase gene included in the 5′-portion of the genome, and structuralgenes included in the 3′-portion of the genome.

Coronavirus Spike (S) protein: A class I fusion glycoprotein initiallysynthesized as a precursor protein. Individual precursor S polypeptidesform a homotrimer and undergo glycosylation within the Golgi apparatusas well as processing to remove the signal peptide, and cleavage by acellular protease to generate separate 51 and S2 polypeptide chains,which remain associated as S1/S2 protomers within the homotrimer and istherefore a trimer of heterodimers. The 51 subunit is distal to thevirus membrane and contains the receptor-binding domain (RBD) thatmediates virus attachment to its host receptor. The S2 subunit containsfusion protein machinery, such as the fusion peptide, two heptad-repeatsequences (HR1 and HR2) and a central helix typical of fusionglycoproteins, a transmembrane domain, and the cytosolic tail domain.

Coronavirus Spike (S) protein prefusion conformation: A structuralconformation adopted by the ectodomain of the coronavirus S proteinfollowing processing into a mature coronavirus S protein in thesecretory system, and prior to triggering of the fusogenic event thatleads to transition of coronavirus S to the postfusion conformation. Thethree-dimensional structure of an exemplary coronavirus S protein(HKU1-CoV) in a prefusion conformation is disclosed herein (seeExample 1) and provided in Kirchdoerfer et al., “Pre-fusion structure ofa human coronavirus spike protein,” Nature, 531: 118-121, 2016(incorporated by reference herein).

A coronavirus S ectodomain trimer “stabilized in a prefusionconformation” comprises one or more amino acid substitutions, deletions,or insertions compared to a native coronavirus S sequence that providefor increased retention of the prefusion conformation compared tocoronavirus S ectodomain trimers formed from a corresponding nativecoronavirus S sequence. The “stabilization” of the prefusionconformation by the one or more amino acid substitutions, deletions, orinsertions can be, for example, energetic stabilization (for example,reducing the energy of the prefusion conformation relative to thepost-fusion open conformation) and/or kinetic stabilization (forexample, reducing the rate of transition from the prefusion conformationto the postfusion conformation). Additionally, stabilization of thecoronavirus S ectodomain trimer in the prefusion conformation caninclude an increase in resistance to denaturation compared to acorresponding native coronavirus S sequence. Methods of determining if acoronavirus S ectodomain trimer is in the prefusion conformation areprovided herein, and include (but are not limited to) negative-stainelectron microscopy and antibody binding assays using aprefusion-conformation-specific antibody.

Degenerate variant: In the context of the present disclosure, a“degenerate variant” refers to a polynucleotide encoding a polypeptidethat includes a sequence that is degenerate as a result of the geneticcode. There are 20 natural amino acids, most of which are specified bymore than one codon. Therefore, all degenerate nucleotide sequencesencoding a peptide are included as long as the amino acid sequence ofthe peptide encoded by the nucleotide sequence is unchanged.

Effective amount: An amount of agent, such as an immunogen, that issufficient to elicit a desired response, such as an immune response in asubject. It is understood that to obtain a protective immune responseagainst an antigen of interest can require multiple administrations of adisclosed immunogen, and/or administration of a disclosed immunogen asthe “prime” in a prime boost protocol wherein the boost immunogen can bedifferent from the prime immunogen. Accordingly, an effective amount ofa disclosed immunogen can be the amount of the immunogen sufficient toelicit a priming immune response in a subject that can be subsequentlyboosted with the same or a different immunogen to elicit a protectiveimmune response.

In one example, a desired response is to inhibit or reduce or preventCoV (such as MERS-CoV) infection. The CoV infection does not need to becompletely eliminated or reduced or prevented for the method to beeffective. For example, administration of an effective amount of theimmunogen can induce an immune response that decreases the CoV infection(for example, as measured by infection of cells, or by number orpercentage of subjects infected by the CoV) by a desired amount, forexample by at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or even at least 100%(elimination or prevention of detectable CoV infection), as compared toa suitable control.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, such that theyelicit a specific immune response, for example, an epitope is the regionof an antigen to which B and/or T cells respond. An antibody can bind toa particular antigenic epitope, such as an epitope on coronavirus Sectodomain, such as a MERS-CoV S ectodomain. Epitopes can be formed bothfrom contiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein.

Expression: Transcription or translation of a nucleic acid sequence. Forexample, a gene is expressed when its DNA is transcribed into an RNA orRNA fragment, which in some examples is processed to become mRNA. A genemay also be expressed when its mRNA is translated into an amino acidsequence, such as a protein or a protein fragment. In a particularexample, a heterologous gene is expressed when it is transcribed into anRNA. In another example, a heterologous gene is expressed when its RNAis translated into an amino acid sequence. The term “expression” is usedherein to denote either transcription or translation. Regulation ofexpression can include controls on transcription, translation, RNAtransport and processing, degradation of intermediary molecules such asmRNA, or through activation, inactivation, compartmentalization ordegradation of specific protein molecules after they are produced.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (ATG) in front of a protein-encoding gene, splicing signal forintrons, maintenance of the correct reading frame of that gene to permitproper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

Expression vector: A vector comprising a recombinant polynucleotidecomprising expression control sequences operatively linked to anucleotide sequence to be expressed. An expression vector comprisessufficient cis-acting elements for expression; other elements forexpression can be supplied by the host cell or in an in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and viruses(e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associatedviruses) that incorporate the recombinant polynucleotide.

Glycosylation site: An amino acid sequence on the surface of apolypeptide, such as a protein, which accommodates the attachment of aglycan. An N-linked glycosylation site is triplet sequence of NX(S/T) inwhich N is asparagine, X is any residues except proline, and (S/T) is aserine or threonine residue. A glycan is a polysaccharide oroligosaccharide. Glycan may also be used to refer to the carbohydrateportion of a glycoconjugate, such as a glycoprotein, glycolipid, or aproteoglycan.

Heterologous: Originating from a different genetic source. A nucleicacid molecule that is heterologous to a cell originated from a geneticsource other than the cell in which it is expressed. In one specific,non-limiting example, a heterologous nucleic acid molecule encoding arecombinant coronavirus S ectodomain is expressed in a cell, such as amammalian cell. Methods for introducing a heterologous nucleic acidmolecule in a cell or organism are well known in the art, for exampletransformation with a nucleic acid, including electroporation,lipofection, particle gun acceleration, and homologous recombination.

Ferritin: A protein that stores iron and releases it in a controlledfashion. The protein is produced by almost all living organisms.Ferritin polypeptides assemble into a globular protein complex of 24protein subunits, and each of the 24 subunits includes a single ferritinpolypeptide. In some examples, ferritin is used to form a nanoparticlepresenting antigens on its surface, for example, a coronavirus Sectodomain trimer.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies.

Immunogen: A compound, composition, or substance (for example, arecombinant coronavirus S ectodomain trimer) that can elicit an immuneresponse in an animal, including compositions that are injected orabsorbed into an animal. Administration of an immunogen to a subject canlead to protective immunity against a pathogen of interest.

Immunogenic composition: A composition comprising a disclosedrecombinant coronavirus S ectodomain trimer that induces a measurableCTL response against the coronavirus, or induces a measurable B cellresponse (such as production of antibodies) against the coronavirus,when administered to a subject. It further refers to isolated nucleicacid molecules and vectors encoding a protomer of a disclosedrecombinant coronavirus S ectodomain trimer that can be used to expressthe protomer (and thus be used to elicit an immune response againstrecombinant coronavirus S ectodomain trimer). For in vivo use, theimmunogenic composition will typically include the recombinantcoronavirus S ectodomain trimer or a nucleic acid molecule encoding aprotomer of the recombinant coronavirus S ectodomain trimer in apharmaceutically acceptable carrier and may also include other agents,such as an adjuvant.

Inhibiting or treating a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as a CoV infection. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition after it has begun to develop. The term“ameliorating,” with reference to a disease or pathological condition,refers to any observable beneficial effect of the treatment. Inhibitinga disease can include preventing or reducing the risk of the disease,such as preventing or reducing the risk of viral infection. Thebeneficial effect can be evidenced, for example, by a delayed onset ofclinical symptoms of the disease in a susceptible subject, a reductionin severity of some or all clinical symptoms of the disease, a slowerprogression of the disease, a reduction in the viral load, animprovement in the overall health or well-being of the subject, or byother parameters that are specific to the particular disease. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

Isolated: An “isolated” biological component has been substantiallyseparated or purified away from other biological components, such asother biological components in which the component naturally occurs,such as other chromosomal and extrachromosomal DNA, RNA, and proteins.Proteins, peptides, nucleic acids, and viruses that have been “isolated”include those purified by standard purification methods.

Isolated does not require absolute purity, and can include protein,peptide, nucleic acid, or virus molecules that are at least 50%isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9%isolated.

Linker and Linked: A bi-functional molecule that can be used to link twomolecules into one contiguous molecule. Non-limiting examples of peptidelinkers include glycine-serine peptide linkers. Unless context indicatesotherwise, reference to “linking” a first polypeptide and a secondpolypeptide, or to two polypeptides “linked” together, or to a firstpolypeptide having a “linkage” to a second polypeptide, refers tocovalent linkage by peptide bond (for example via a peptide linker) suchthat the first and second polypeptides form a contiguous polypeptidechain. If a peptide linker is involved, the covalent linkage of thefirst and second polypeptides can be to the N- and C-termini of thepeptide linker Typically, such linkage is accomplished using molecularbiology techniques to genetically manipulate DNA encoding the firstpolypeptide linked to the second polypeptide by the peptide linker.

Native protein, sequence, or disulfide bond: A polypeptide, sequence ordisulfide bond that has not been modified, for example, by selectivemutation. For example, selective mutation to focus the antigenicity ofthe antigen to a target epitope, or to introduce a disulfide bond into aprotein that does not occur in the native protein. Native protein ornative sequence are also referred to as wild-type protein or wild-typesequence. A non-native disulfide bond is a disulfide bond that is notpresent in a native protein, for example, a disulfide bond that forms ina protein due to introduction of one or more cysteine residues into theprotein by genetic engineering.

Nucleic acid molecule: A polymeric form of nucleotides, which mayinclude both sense and anti-sense strands of RNA, cDNA, genomic DNA, andsynthetic forms and mixed polymers of the above. A nucleotide refers toa ribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide. The term “nucleic acid molecule” as used herein issynonymous with “nucleic acid” and “polynucleotide.” A nucleic acidmolecule is usually at least 10 bases in length, unless otherwisespecified. The term includes single- and double-stranded forms of DNA. Apolynucleotide may include either or both naturally occurring andmodified nucleotides linked together by naturally occurring and/ornon-naturally occurring nucleotide linkages. “cDNA” refers to a DNA thatis complementary or identical to an mRNA, in either single stranded ordouble stranded form. “Encoding” refers to the inherent property ofspecific sequences of nucleotides in a polynucleotide, such as a gene, acDNA, or an mRNA, to serve as templates for synthesis of other polymersand macromolecules in biological processes having either a definedsequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a definedsequence of amino acids and the biological properties resultingtherefrom.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked nucleic acid sequences arecontiguous and, where necessary to join two protein-coding regions, inthe same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the disclosed immunogens.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions (such as immunogenic compositions) to be administered cancontain minor amounts of non-toxic auxiliary substances, such as wettingor emulsifying agents, preservatives, and pH buffering agents and thelike, for example sodium acetate or sorbitan monolaurate. In particularembodiments, suitable for administration to a subject the carrier may besterile, and/or suspended or otherwise contained in a unit dosage formcontaining one or more measured doses of the composition suitable toinduce the desired immune response. It may also be accompanied bymedications for its use for treatment purposes. The unit dosage form maybe, for example, in a sealed vial that contains sterile contents or asyringe for injection into a subject, or lyophilized for subsequentsolubilization and administration or in a solid or controlled releasedosage.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). “Polypeptide” applies to amino acid polymers includingnaturally occurring amino acid polymers and non-naturally occurringamino acid polymer as well as in which one or more amino acid residue isa non-natural amino acid, for example, an artificial chemical mimetic ofa corresponding naturally occurring amino acid. A “residue” refers to anamino acid or amino acid mimetic incorporated in a polypeptide by anamide bond or amide bond mimetic. A polypeptide has an amino terminal(N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide”is used interchangeably with peptide or protein, and is used herein torefer to a polymer of amino acid residues.

Prime-boost vaccination: An immunotherapy including administration of afirst immunogenic composition (the primary vaccine) followed byadministration of a second immunogenic composition (the booster vaccine)to a subject to induce an immune response. The priming vaccine and/orthe booster vaccine include a vector (such as a viral vector, RNA, orDNA vector) expressing the antigen to which the immune response isdirected. The booster vaccine is administered to the subject after thepriming vaccine; a suitable time interval between administration of thepriming vaccine and the booster vaccine, and examples of such timeframesare disclosed herein. In some embodiments, the priming vaccine, thebooster vaccine, or both primer vaccine and the booster vaccineadditionally include an adjuvant. In one non-limiting example, thepriming vaccine is a DNA-based vaccine (or other vaccine based on genedelivery), and the booster vaccine is a protein subunit or proteinnanoparticle based vaccine.

Protein nanoparticle: A multi-subunit, self-assembling, protein-basedpolyhedron shaped structure. The subunits are each composed of proteins(for example a glycosylated polypeptide), and, optionally of single ormultiple features of the following: nucleic acids, prosthetic groups,organic and inorganic compounds. In some embodiments, protomers of thedisclosed trimeric spike proteins can be fused to the subunits of theprotein nanoparticles to provide multiple copies of the trimeric spikeon each protein nanoparticle. Non-limiting examples of proteinnanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y. Int.J. Mol. Sci., 12:5406-5421, 2011, incorporated by reference herein),encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct. andMol. Biol., 15:939-947, 2008, incorporated by reference herein), SulfurOxygenase Reductase (SOR) nanoparticles (see, e.g., Urich et al.,Science, 311:996-1000, 2006, incorporated by reference herein), lumazinesynthase nanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306:1099-1114, 2001), and pyruvate dehydrogenase nanoparticles (see, e.g.,Izard et al., PNAS 96: 1240-1245, 1999, incorporated by referenceherein). Ferritin, encapsulin, SOR, lumazine synthase, and pyruvatedehydrogenase are monomeric proteins that self-assemble into a globularprotein complexes that in some cases consists of 24, 60, 24, 60, and 60protein subunits, respectively. Additional protein nanoparticlestructures are described by Heinze et al., J Phys Chem B.,120(26):5945-52, 2016; Hsia et al., Nature, 535(7610):136-9, 2016; andKing et al., Nature, 510(7503):103-8, 2014; each of which isincorporated by reference herein.

Recombinant: A recombinant nucleic acid molecule is one that has asequence that is not naturally occurring, for example, includes one ormore nucleic acid substitutions, deletions or insertions, and/or has asequence that is made by an artificial combination of two otherwiseseparated segments of sequence. This artificial combination can beaccomplished by chemical synthesis or, more commonly, by the artificialmanipulation of isolated segments of nucleic acids, for example, bygenetic engineering techniques. A recombinant virus is one that includesa genome that includes a recombinant nucleic acid molecule. Arecombinant protein is one that has a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo otherwise separated segments of sequence. In several embodiments, arecombinant protein is encoded by a heterologous (for example,recombinant) nucleic acid that has been introduced into a host cell,such as a bacterial or eukaryotic cell, or into the genome of arecombinant virus.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity; the higher the percentage, themore similar the two sequences are. Homologs, orthologs, or variants ofa polypeptide will possess a relatively high degree of sequence identitywhen aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

Homologs and variants of a polypeptide (such as a coronavirus Sectodomain) are typically characterized by possession of at least about75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity counted over the full lengthalignment with the amino acid sequence of interest. Proteins with evengreater similarity to the reference sequences will show increasingpercentage identities when assessed by this method, such as at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% sequence identity. When less than the entire sequence is beingcompared for sequence identity, homologs and variants will typicallypossess at least 80% sequence identity over short windows of 10-20 aminoacids, and may possess sequence identities of at least 85% or at least90% or 95% depending on their similarity to the reference sequence.Methods for determining sequence identity over such short windows areavailable at the NCBI website on the internet.

As used herein, reference to “at least 90% identity” or similar languagerefers to “at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or even 100% identity” to a specified reference sequence.

Signal Peptide: A short amino acid sequence (e.g., approximately 10-35amino acids in length) that directs newly synthesized secretory ormembrane proteins to and through membranes (for example, the endoplasmicreticulum membrane). Signal peptides are typically located at theN-terminus of a polypeptide and are removed by signal peptidases. Signalpeptide sequences typically contain three common structural features: anN-terminal polar basic region (n-region), a hydrophobic core, and ahydrophilic c-region).

Single chain coronavirus S ectodomain: A recombinant coronavirus Sectodomain including the coronavirus S₁ and S₂ proteins in a singlecontiguous polypeptide chain. Single chain coronavirus S ectodomain cantrimerize to form a coronavirus S ectodomain trimer. A singlecoronavirus S ectodomain includes mutations to prevent protease cleavageat the S₁/S₂ cleavage site and the S₂′ cleavage site in the Sectodomain. Therefore, when produced in cells, the S polypeptide is notcleaved into separate S₁ and S₂ polypeptide chains.

Soluble protein: A protein capable of dissolving in aqueous liquid atroom temperature and remaining dissolved. The solubility of a proteinmay change depending on the concentration of the protein in thewater-based liquid, the buffering condition of the liquid, theconcentration of other solutes in the liquid, for example salt andprotein concentrations, and the heat of the liquid. In severalembodiments, a soluble protein is one that dissolves to a concentrationof at least 0.5 mg/ml in phosphate buffered saline (pH 7.4) at roomtemperature and remains dissolved for at least 48 hours.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals, such as non-human primates, pigs,camels, bats, sheep, cows, dogs, cats, rodents, and the like. In anexample, a subject is a human. In a particular example, the subject is acamel or a bat. The subject can be a domestic animal (such as a dog or acat) or a farm animal (such as a cow or a pig). In an additionalexample, a subject is selected that is in need of inhibiting of acoronavirus infection, such as a SARS-CoV or MERS-CoV infection. Forexample, the subject is either uninfected and at risk of the coronavirusinfection or is infected and in need of treatment.

T4 Fibritin trimerization domain: Also referred to as a “foldon” domain,the T4 Fibritin trimerization domain comprises an amino acid sequencethat naturally forms a trimeric structure. In some examples, a T4Fibritin trimerization domain can be linked to the C-terminus of adisclosed recombinant coronavirus S protein ectodomain. In one example,a T4 Fibritin trimerization domain comprises the amino acid sequence setforth as (GYIPEAPRDGQAYVRKDGEWVLLSTF (SEQ ID NO: 22). In someembodiments, a protease cleavage site (such as a thrombin cleavage site)can be included between the C-terminus of the recombinant coronavirusectodomain and the T4 Fibritin trimerization domain to facilitateremoval of the trimerization domain as needed, for example, followingexpression and purification of the recombinant coronavirus S ectodomain.

Transmembrane domain: An amino acid sequence that inserts into a lipidbilayer, such as the lipid bilayer of a cell or virus or virus-likeparticle. A transmembrane domain can be used to anchor an antigen to amembrane. In some examples a transmembrane domain is a coronavirus Stransmembrane domain, such as a MERS-CoV or SARS-CoV S transmembranedomain.

Vaccine: A pharmaceutical composition that induces a prophylactic ortherapeutic immune response in a subject. In some cases, the immuneresponse is a protective immune response. Typically, a vaccine inducesan antigen-specific immune response to an antigen of a pathogen, forexample a viral pathogen, or to a cellular constituent correlated with apathological condition. A vaccine may include a polynucleotide (such asa nucleic acid encoding a disclosed antigen), a peptide or polypeptide(such as a disclosed antigen), a virus, a cell or one or more cellularconstituents. In a non-limiting example, a vaccine induces an immuneresponse that reduces the severity of the symptoms associated with acoronavirus infection (such as a SARS-CoV or MERS-CoV infection) and/ordecreases the viral load compared to a control. In another non-limitingexample, a vaccine induces an immune response that reduces and/orprevents a coronavirus infection (such as a SARS-CoV or MERS-CoVinfection) compared to a control.

Vector: An entity containing a DNA or RNA molecule bearing a promoter(s)that is operationally linked to the coding sequence of an antigen(s) ofinterest and can express the coding sequence. Non-limiting examplesinclude a naked or packaged (lipid and/or protein) DNA, a naked orpackaged RNA, a subcomponent of a virus or bacterium or othermicroorganism that may be replication-incompetent, or a virus orbacterium or other microorganism that may be replication-competent. Avector is sometimes referred to as a construct. Recombinant DNA vectorsare vectors having recombinant DNA. A vector can include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector can also include one or more selectable markergenes and other genetic elements known in the art. Viral vectors arerecombinant nucleic acid vectors having at least some nucleic acidsequences derived from one or more viruses.

Virus-like particle (VLP): A non-replicating, viral shell, derived fromany of several viruses. VLPs are generally composed of one or more viralproteins, such as, but not limited to, those proteins referred to ascapsid, coat, shell, surface and/or envelope proteins, orparticle-forming polypeptides derived from these proteins. VLPs can formspontaneously upon recombinant expression of the protein in anappropriate expression system. Methods for producing particular VLPs areknown in the art. The presence of VLPs following recombinant expressionof viral proteins can be detected using conventional techniques known inthe art, such as by electron microscopy, biophysical characterization,and the like. Further, VLPs can be isolated by known techniques, e.g.,density gradient centrifugation and identified by characteristic densitybanding. See, for example, Baker et al. (1991) Biophys. J. 60:1445-1456;and Hagensee et al. (1994) J. Virol. 68:4503-4505; Vincente, J InvertebrPathol., 2011; Schneider-Ohrum and Ross, Curr. Top. Microbiol. Immunol.,354: 53073, 2012).

II. Immunogens

Disclosed herein are recombinant coronavirus (such as alphacoronavirusor betacoronavirus) S ectodomain trimers comprising protomers comprisingone or more proline substitution(s). The proline substitutions inhibit aconformational change in the S protein from the prefusion conformationto the postfusion conformation, and therefore stabilize the S ectodomaintrimer in the prefusion conformation. In some embodiments, therecombinant coronavirus (such as alphacoronavirus or betacoronavirus) Sectodomain trimer comprises protomers comprising one or more (such astwo) proline substitutions at or near the boundary between a HR1 domainand a central helix domain of the protomers. In some embodiments, theone or more (such as two, for example two consecutive) prolinesubstitutions that stabilize the protomers of the S ectodomain in theprefusion conformation are located between a position 15 amino acidsN-terminal of a C-terminal residue of the HR1 and a position 5 aminoacids C-terminal of a N-terminal residue of the central helix. Exemplaryembodiments are shown to produce a superior immune response in an animalmodel compared to corresponding coronavirus S ectodomain trimers thatare not stabilized in the prefusion conformation.

In some embodiments, the recombinant S ectodomain trimer comprisesrecombinant S ectodomain protomers from an alphacoronavirus, such asNL63-CoV or 229E-CoV, that have been mutated to include the one or moreproline substitutions for stabilization in the prefusion conformation.In some embodiments, the recombinant S ectodomain trimers comprise Sectodomain protomers from a betacoronavirus, such as OC43-CoV, SARS-CoV,MERS-CoV, HKU1-CoV, WIV1-CoV, mouse hepatitis virus (MHV), or HKU9-CoV,that have been mutated to include the one or more proline substitutionsfor stabilization in the prefusion conformation. Additional descriptionis provided below.

A. MERS-CoV

In some embodiments, the immunogen comprises a recombinant MERS-CoV Sectodomain trimer comprising protomers comprising one or more (such astwo, for example two consecutive) proline substitutions at or near theboundary between a HR1 domain and a central helix domain that stabilizethe S ectodomain trimer in the prefusion conformation. In some suchembodiments, the one or more (such as two, for example two consecutive)proline substitutions that stabilize the S ectodomain in the prefusionconformation are located between a position 15 amino acids N-terminal ofa C-terminal residue of the HR1 and a position 5 amino acids C-terminalof a N-terminal residue of the central helix.

In some embodiments, the one or more (such as two, for example twoconsecutive) proline substitutions that stabilize the MERS-CoV Sectodomain trimer in the prefusion conformation are located betweenresidues 1050 to 1069 (such as between residues 1053 to 1063, or betweenresidues 1058 to 1063) of the S ectodomain protomers in the trimer. Insome embodiments, the MERS-CoV S ectodomain trimer is stabilized in theprefusion conformation by one or two of: L1058P, D1059P, V1060P, andL1061P substitutions in the S ectodomain protomers in the trimer. Insome embodiments, the MERS-CoV S ectodomain trimer is stabilized in theprefusion conformation by V1060P and L1061P substitutions (“2P”) in theS ectodomain protomers in the trimer. The amino acid numbering forMERS-CoV S proteins is with reference to the MERS-CoV S sequenceprovided as SEQ ID NO: 1.

In some embodiments, the recombinant MERS-CoV S ectodomain trimerstabilized in the prefusion conformation comprises protomers ofsingle-chain S ectodomains comprising mutations to the S1/S2 and/or S2′protease cleavage sites to prevent protease cleavage at these sites.Non-limiting examples of such mutations include 748-RSVR-751 (residues748-751 of SEQ ID NO: 1) to 748-ASVG-751 (residues 748-751 of SEQ ID NO:3) substitutions to inhibit cleavage at the S1/S2 cleavage site, and884-RSAR-887 (residues 884-887 of SEQ ID NO: 1) to 884-GSAG-887(residues 884-887 of SEQ ID NO: 3) substitutions to inhibit cleavage atthe S2′ site.

In some embodiments, the recombinant MERS-CoV S ectodomain trimercomprising protomers stabilized in the prefusion conformation by the oneor more proline substitutions (such as V1060P and L1061P substitutions)comprises additional modifications for stabilization in the prefusionconformation. In some embodiments, the recombinant MERS-CoV S ectodomaintrimer comprising protomers stabilized in the prefusion conformation bythe one or more proline substitutions (such as V1060P and L1061Psubstitutions) further comprises cavity filling substitutions tostabilize the S ectodomain the prefusion conformation, such as one of:N1072F and A1083I; N1072F and L1086F; N1072F and V1087I; N1072F andE1090I; T1076F and A1083I; T1076F and L1086F; T1076F and V1087I; T1076Fand E1090I; T1076I and A1083I; T1076I and L1086F; T1076I and V1087I;T1076I and E1090I; A1018V; or A1018I.

In some embodiments, the recombinant MERS-CoV S ectodomain trimerstabilized in the prefusion conformation by the one or more prolinesubstitutions (such as V1060P and L1061P substitutions) furthercomprises a repacking substitution to stabilize the S ectodomain theprefusion conformation, such as one of: E793M and K1102F; E793M, K1102F,and H1138F; D1068M and R1069W; A1083L; A1083L and V1087I; A1083L, V1087,and E1090L; A834L and Q1084M; Q1066M; 5454F; R921W; S612F and G1052F; orP476V, T477A, and R1057W.

In some embodiments, the recombinant MERS-CoV S ectodomain trimerstabilized in the prefusion conformation by the one or more prolinesubstitutions (such as V1060P and L1061P substitutions) furthercomprises one of A1083S, E1090I, Q1097I, D1101F, or A653W to stabilizethe S ectodomain the prefusion conformation.

In some embodiments, the recombinant MERS-CoV S ectodomain trimerstabilized in the prefusion conformation by the one or more prolinesubstitutions (such as V1060P and L1061P substitutions) furthercomprises a non-native disulfide bond formed between cysteinesintroduced by one of: T63C and V631C; T63C and Q638C; Q733C and D940C;S676C and D910C; V1087C (which forms a disulfide bond with a cysteinepresent in the native sequence); A432C and L1058C; or A432C and D1059Cto stabilize the S ectodomain the prefusion conformation.

In some embodiments, the recombinant MERS-CoV S ectodomain trimerstabilized in the prefusion conformation by the one or more prolinesubstitutions (such as V1060P and L1061P substitutions) furthercomprises an additional proline substitution to stabilize the Sectodomain the prefusion conformation, such as one of: K801P; V802P;T803P; V804P; S919P; A920P; A968P; A969P; I970P; F972P; A973P; T1014P;N1042P; T1043P; F1044P; G1045P; A1046P; I1047P; or A1049P.

Any of the substitutions described above can be combined in the MERS-CoVS ectodomain trimer, as long as the trimer is stabilized in theprefusion conformation and can be used to generate a neutralizing immuneresponse to MERS-CoV in a subject.

With reference to the MERS-CoV S protein sequence provided as SEQ ID NO:1, the ectodomain of the MERS-CoV S protein includes about residues18-1291. Residues 1-17 are the signal peptide, which is removed duringcellular processing. The S1/S2 cleavage site is located at aboutposition 751/752. The S2′ cleavage site is located at about position887/888. The HR1 is located at about residues 989-1057. The centralhelix is located at about residues 1062-1103. The HR2 is located atabout 1246-1277. The C-terminal end of the S2 ectodomain is located atabout residue 1291. In some embodiments, the protomers of theprefusion-stabilized MERS-CoV S ectodomain trimer can have a C-terminalresidue (which can be linked to a trimerization domain, or atransmembrane domain, for example) of the C-terminal residue of the HR2(e.g., position 1277), or the ectodomain (e.g., position 1291) or fromone of positions 1277-1291. The position numbering of the S protein mayvary between MERS-CoV stains, but the sequences can be aligned todetermine relevant structural domains and cleavage sites. It will beappreciated that a few residues (such as up to 10) on the N andC-terminal ends of the ectodomain can be removed or modified in thedisclosed immunogens without decreasing the utility of the S ectodomaintrimer as an immunogen.

Exemplary MERS-CoV S protein sequences are provided below. Any of theMERS-CoV S protein mutations (such as V1060P and L1061P, and/ormodifications to generate a single chain) can be incorporated in theMERS-CoV S protein sequences.

An exemplary sequence of MERS-CoV S protein including the ectodomain andTM and CT domains) England1 strain is provided as SEQ ID NO: 1:

MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFHKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWPWYIWLGFIAGLVALALCVFFILCCTGCGTNCMGKLKCNRCCDRYEEYDLEPHKV HVH

An exemplary sequence of MERS-CoV S ectodomain England1 strain includingV1060P and L1061P substitutions is provided as SEQ ID NO: 2:

MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFHKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDPPEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTY

An exemplary sequence of MERS-CoV S ectodomain England1 strain includingV1060P and L1061P substitutions and 748-RSVR-751 (residues 748-751 ofSEQ ID NO: 1) to 748-ASVG-751 (residues 748-751 of SEQ ID NO: 3)substitutions to remove the S1/S2 cleavage site is provided as SEQ IDNO: 3:

MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPASVGSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFHKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDPPEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTY

An exemplary sequence of MERS-CoV S ectodomain England1 strain includingV1060P and L1061P substitutions and 748-RSVR-751 (residues 748-751 ofSEQ ID NO: 1) to 748-ASVG-751 (residues 748-751 of SEQ ID NO: 3) and884-RSAR-887 (residues 884-887 of SEQ ID NO: 1) to 884-GSAG-887(residues 884-887 of SEQ ID NO: 3) substitutions to remove the S1/S2cleavage site and the S2′ cleavage site is provided as SEQ ID NO: 4:

MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPASVGSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSGSAGSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFHKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDPPEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTY

A C-terminal trimerization domain can be added to the protomers of theMERS-CoV S ectodomains trimer to promote trimerization of theectodomain.

An exemplary sequence of MERS-CoV S ectodomain England1 strain includingV1060P and L1061P substitutions and 748-RSVR-751 (residues 748-751 ofSEQ ID NO: 1) to 748-ASVG-751 (residues 748-751 of SEQ ID NO: 3)substitutions to remove the S1/S2 cleavage site, and a T4 fibritintrimerization domain is provided as SEQ ID NO: 28:

MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPASVGSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFHKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDPPEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYGGYIPEAPR DGQAYVRKDGEWVLLSTF

An exemplary sequence of MERS-CoV S ectodomain England1 strain includingV1060P and L1061P substitutions and 748-RSVR-751 to 748-ASVG-751 and884-RSAR-887 (residues 884-887 of SEQ ID NO: 1) to 884-GSAG-887(residues 884-887 of SEQ ID NO: 3) substitutions to remove the S1/S2cleavage site and the S2′ cleavage site, and a T4 fibritin trimerizationdomain is provided as SEQ ID NO: 29:

MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPASVGSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSGSAGSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFHKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDPPEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYGGYIPEAPR DGQAYVRKDGEWVLLSTF

In some embodiments, the recombinant MERS-CoV S ectodomain trimercomprises protomers comprising the ectodomain sequence of any one of SEQID NOs: 2-4 and 29. In some embodiments, the recombinant MERS-CoV Sectodomain trimer comprises protomers comprising residues 18-1291 of anyone of SEQ ID NOs: 2-4 or residues 18-1318 of SEQ ID NO: 29. In someembodiments, the recombinant MERS-CoV S ectodomain trimer comprisesprotomers comprising an ectodomain sequence at least 90% identical tothe ectodomain sequence of any one of SEQ ID NOs: 2-4, wherein theMERS-CoV S ectodomain trimer is stabilized in the prefusion conformationand comprises the “2P” substitution and/or modifications to remove theS1/S2 cleavage site and the S2′ cleavage site of the protomers. In someembodiments, the recombinant MERS-CoV S ectodomain trimer comprisesprotomers comprising an amino acid sequence at least 90% identical toresidues 18-1291 of any one of SEQ ID NOs: 2-4 or residues 18-1318 ofSEQ ID NO: 29, wherein the MERS-CoV S ectodomain trimer is stabilized inthe prefusion conformation and comprises the “2P” substitution and/ormodifications to remove the S1/S2 cleavage site and the S2′ cleavagesite of the protomers.

B. SARS-CoV

In some embodiments, the immunogen comprises a recombinant SARS-CoV Sectodomain trimer comprising protomers comprising one or more (such astwo, for example two consecutive) proline substitutions at or near theboundary between a HR1 domain and a central helix domain that stabilizethe S ectodomain trimer in the prefusion conformation. In some suchembodiments, the one or more (such as two, for example two consecutive)proline substitutions that stabilize the S ectodomain in the prefusionconformation are located between a position 15 amino acids N-terminal ofa C-terminal residue of the HR1 and a position 5 amino acids C-terminalof a N-terminal residue of the central helix.

In some embodiments, the one or more (such as two, for example twoconsecutive) proline substitutions that stabilize the SARS-CoV Sectodomain trimer in the prefusion conformation are located betweenresidues 951 to 971 (such as between residues 961 to 971 or betweenresidues 966 to 971) of the S ectodomain protomers in the trimer. Insome embodiments, the SARS-CoV S ectodomain trimer is stabilized in theprefusion conformation by K968P and V969P substitutions (“2P”) in the Sectodomain protomers in the trimer. The amino acid numbering forSARS-CoV S proteins is with reference to the SARS-CoV S sequenceprovided as SEQ ID NO: 6.

In some embodiments, the recombinant SARS-CoV S ectodomain trimerstabilized in the prefusion conformation comprises single-chain Sectodomain protomers comprising mutations to the S1/S2 and/or S2′protease cleavage sites to prevent protease cleavage at these sites.

In some embodiments, the protomers of the recombinant SARS-CoV Sectodomain trimer stabilized in the prefusion conformation by the one ormore proline substitutions (such as K968P and V969P substitutions)comprises additional modifications for stabilization in the prefusionconformation.

With reference to the SARS-CoV S protein sequence provided as SEQ ID NO:6, the ectodomain of the SARS-CoV S protein includes about residues14-1190. Residues 1-13 are the signal peptide, which is removed duringcellular processing. The S1/S2 cleavage site is located at position667/668 or 678/679. The S2′ cleavage site is located at about position797/798. The HR1 is located at about residues 897-965. The central helixis located at about residues 970-1011. The HR2 is located at about1145-1176. The C-terminal end of the S2 ectodomain is located at aboutresidue 1190. In some embodiments, the protomers of theprefusion-stabilized SARS-CoV S ectodomain trimer can have a C-terminalresidue (which can be linked to a trimerization domain, or atransmembrane domain, for example) of the C-terminal residue of the HR2(e.g., position 1176), or the ectodomain (e.g., position 1190), or fromone of positions 1176-1190. The position numbering of the S protein mayvary between SARS-CoV stains, but the sequences can be aligned todetermine relevant structural domains and cleavage sites. It will beappreciated that a few residues (such as up to 10) on the N andC-terminal ends of the ectodomain can be removed or modified in thedisclosed immunogens without decreasing the utility of the S ectodomaintrimer as an immunogen.

Exemplary SARS-CoV S protein sequences are provided below. The prefusionstabilizing substitutions disclosed herein (and other modifications,such as substitutions to generate a single chain) can be incorporatedinto SARS-CoV S protein sequences.

An exemplary sequence of SARS-CoV S protein (including the ectodomainand TM and CT domains) is provided as SEQ ID NO: 6 (GenBank GI:30795145, incorporated by reference herein):

MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGV KLHYT

An exemplary sequence of SARS-CoV S ectodomain (TOR2 strain) including adouble proline substitution for stabilization in the prefusionconformation is provided as SEQ ID NO: 7:

MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

A C-terminal trimerization domain can be added to the protomers of theSARS-CoV S ectodomains trimer to promote trimerization of theectodomain.

An exemplary sequence of SARS-CoV S ectodomain (TOR2 strain) including adouble proline substitution for stabilization in the prefusionconformation, and a T4 fibritin trimerization domain is provided as SEQID NO: 30:

MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGGYIPEAPRD GQAYVRKDGEWVLLSTF

In some embodiments, the recombinant SARS-CoV S ectodomain trimercomprises protomers comprising the ectodomain sequence of SEQ ID NO: 7.In some embodiments, the recombinant SARS-CoV S ectodomain trimercomprises protomers comprising residues 14-1190 of SEQ ID NO: 7 orresidues 14-1217 of SEQ ID NO: 30. In some embodiments, the recombinantSARS-CoV S ectodomain trimer comprises protomers comprising anectodomain sequence at least 90% identical to the ectodomain sequence ofSEQ ID NO: 7, wherein the SARS-CoV S ectodomain trimer is stabilized inthe prefusion conformation and comprises the “2P” substitution and/ormodifications to remove the S1/S2 cleavage site and the S2′ cleavagesite of the protomers. In some embodiments, the recombinant SARS-CoV Sectodomain trimer comprises protomers comprising an amino acid sequenceat least 90% identical to residues 14-1190 of SEQ ID NO: 7, wherein theSARS-CoV S ectodomain trimer is stabilized in the prefusion conformationand comprises the “2P” substitution and/or modifications to remove theS1/S2 cleavage site and the S2′ cleavage site of the protomers.

C. HKU1-CoV

In some embodiments, the immunogen comprises a recombinant HKU1-CoV Sectodomain trimer comprising protomers comprising one or more (such astwo, for example two consecutive) proline substitutions at or near theboundary between a HR1 domain and a central helix domain that stabilizethe S ectodomain trimer in the prefusion conformation. In some suchembodiments, the one or more (such as two, for example two consecutive)proline substitutions that stabilize the S ectodomain in the prefusionconformation are located between a position 15 amino acids N-terminal ofa C-terminal residue of the HR1 and a position 5 amino acids C-terminalof a N-terminal residue of the central helix.

In some embodiments, the one or more (such as two, for example twoconsecutive) proline substitutions that stabilize the HKU1-CoV Sectodomain trimer in the prefusion conformation are located betweenresidues 1050 to 1070 (such as between residues 1060 to 1070 or betweenresidues 1065 to 1070) of the S ectodomain protomers in the trimer. Insome embodiments, the HKU1-CoV S ectodomain trimer is stabilized in theprefusion conformation by N1067P and L1068P substitutions (“2P”) in theS ectodomain protomers in the trimer. The amino acid numbering forHKU1-CoV S proteins is with reference to the HKU1-CoV S sequenceprovided as SEQ ID NO: 7.

In some embodiments, the recombinant HKU1-CoV S ectodomain trimerstabilized in the prefusion conformation comprises single-chain Sectodomain protomers comprising mutations to the S1/S2 and/or S2′protease cleavage sites to prevent protease cleavage at these sites.

In some embodiments, the protomers of the recombinant HKU1-CoV Sectodomain trimer stabilized in the prefusion conformation by the one ormore proline substitutions (such as N1067P and L1068P substitutions)comprise additional modifications for stabilization in the prefusionconformation.

With reference to the HKU1-CoV S protein sequence provided as SEQ ID NO:8, the ectodomain of the HKU1-CoV S protein includes about residues14-1290. Residues 1-13 are the signal peptide, which is removed duringcellular processing. The S1/S2 cleavage site is located at aboutposition 756/757. The S2′ cleavage site is located at about position900/901. The HR1 is located at about residues 996-1064. The centralhelix is located at about residues 1069-1110. The HR2 is located atabout 1245-1276. The C-terminal end of the S2 ectodomain is located atabout residue 1290. In some embodiments, the protomers of theprefusion-stabilized HKU1-CoV S ectodomain trimer can have a C-terminalresidue (which can be linked to a trimerization domain, or atransmembrane domain, for example) of the C-terminal residue of the HR2(e.g., position 1276), or the ectodomain (e.g., position 1290), or fromone of positions 1276-1290. The position numbering of the S protein mayvary between HKU1-CoV stains, but the sequences can be aligned todetermine relevant structural domains and cleavage sites. It will beappreciated that a few residues (such as up to 10) on the N andC-terminal ends of the ectodomain can be removed or modified in thedisclosed immunogens without decreasing the utility of the S ectodomaintrimer as an immunogen.

Exemplary HKU1-CoV S protein sequences are provided below. The prefusionstabilizing substitutions disclosed herein (and other modifications,such as substitutions to generate a single chain) can be incorporatedinto HKU1-CoV S protein sequences.

An exemplary sequence of HKU1-CoV S protein (including the ectodomainand TM and CT domains) is provided as SEQ ID NO: 8 (GenBank GI:123867264, incorporated by reference herein):

MFLIIFILPTTLAVIGDFNCTNSFINDYNKTIPRISEDVVDVSLGLGTYYVLNRVYLNTTLLFTGYFPKSGANFRDLALKGSIYLSTLWYKPPFLSDFNNGIFSKVKNTKLYVNNTLYSEFSTIVIGSVFVNTSYTIVVQPHNGILEITACQYTMCEYPHTVCKSKGSIRNESWHIDSSEPLCLFKKNFTYNVSADWLYFHFYQERGVFYAYYADVGMPTTFLFSLYLGTILSHYYVMPLTCNAISSNTDNETLEYWVTPLSRRQYLLNFDEHGVITNAVDCSSSFLSEIQCKTQSFAPNTGVYDLSGFTVKPVATVYRRIPNLPDCDIDNWLNNVSVPSPLNWERRIFSNCNFNLSTLLRLVHVDSFSCNNLDKSKIFGSCFNSITVDKFAIPNRRRDDLQLGSSGFLQSSNYKIDISSSSCQLYYSLPLVNVTINNFNPSSWNRRYGFGSFNLSSYDVVYSDHCFSVNSDFCPCADPSVVNSCAKSKPPSAICPAGTKYRHCDLDTTLYVKNWCRCSCLPDPISTYSPNTCPQKKVVVGIGEHCPGLGINEEKCGTQLNHSSCFCSPDAFLGWSFDSCISNNRCNIFSNFIFNGINSGTTCSNDLLYSNTEISTGVCVNYDLYGITGQGIFKEVSAAYYNNWQNLLYDSNGNIIGFKDFLTNKTYTILPCYSGRVSAAFYQNSSSPALLYRNLKCSYVLNNISFISQPFYFDSYLGCVLNAVNLTSYSVSSCDLRMGSGFCIDYALPSSRRKRRGISSPYRFVTFEPFNVSFVNDSVETVGGLFEIQIPTNFTIAGHEEFIQTSSPKVTIDCSAFVCSNYAACHDLLSEYGTFCDNINSILNEVNDLLDITQLQVANALMQGVTLSSNLNTNLHSDVDNIDFKSLLGCLGSQCGSSSRSLLEDLLFNKVKLSDVGFVEAYNNCTGGSEIRDLLCVQSFNGIKVLPPILSETQISGYTTAATVAAMFPPWSAAAGVPFSLNVQYRINGLGVTMDVLNKNQKLIANAFNKALLSIQNGFTATNSALAKIQSVVNANAQALNSLLQQLFNKFGAISSSLQEILSRLDNLEAQVQIDRLINGRLTALNAYVSQQLSDITLIKAGASRAIEKVNECVKSQSPRINFCGNGNHILSLVQNAPYGLLFIHFSYKPTSFKTVLVSPGLCLSGDRGIAPKQGYFIKQNDSWMFTGSSYYYPEPISDKNVVFMNSCSVNFTKAPFIYLNNSIPNLSDFEAELSLWFKNHTSIAPNLTFNSHINATFLDLYYEMNVIQESIKSLNSSFINLKEIGTYEMYVKWPWYIWLLIVILFIIFLMILFFICCCTGCGSACFSKCHNCCDEYGGHNDFVIKASHD D

An exemplary sequence of HKU1-CoV S ectodomain including a doubleproline substitution for stabilization in the prefusion conformation isprovided as (SEQ ID NO: 9, which also includes mutations to eliminatethe S1/S2 cleavage site:

MFLIIFILPTTLAVIGDFNCTNSFINDYNKTIPRISEDVVDVSLGLGTYYVLNRVYLNTTLLFTGYFPKSGANFRDLALKGSIYLSTLWYKPPFLSDFNNGIFSKVKNTKLYVNNTLYSEFSTIVIGSVFVNTSYTIVVQPHNGILEITACQYTMCEYPHTVCKSKGSIRNESWHIDSSEPLCLFKKNFTYNVSADWLYFHFYQERGVFYAYYADVGMPTTFLFSLYLGTILSHYYVMPLTCNAISSNTDNETLEYWVTPLSRRQYLLNFDEHGVITNAVDCSSSFLSEIQCKTQSFAPNTGVYDLSGFTVKPVATVYRRIPNLPDCDIDNWLNNVSVPSPLNWERRIFSNCNFNLSTLLRLVHVDSFSCNNLDKSKIFGSCFNSITVDKFAIPNRRRDDLQLGSSGFLQSSNYKIDISSSSCQLYYSLPLVNVTINNFNPSSWNRRYGFGSFNLSSYDVVYSDHCFSVNSDFCPCADPSVVNSCAKSKPPSAICPAGTKYRHCDLDTTLYVKNWCRCSCLPDPISTYSPNTCPQKKVVVGIGEHCPGLGINEEKCGTQLNHSSCFCSPDAFLGWSFDSCISNNRCNIFSNFIFNGINSGTTCSNDLLYSNTEISTGVCVNYDLYGITGQGIFKEVSAAYYNNWQNLLYDSNGNIIGFKDFLTNKTYTILPCYSGRVSAAFYQNSSSPALLYRNLKCSYVLNNISFISQPFYFDSYLGCVLNAVNLTSYSVSSCDLRMGSGFCIDYALPSSGGSGSGISSPYRFVTFEPFNVSFVNDSVETVGGLFEIQIPTNFTIAGHEEFIQTSSPKVTIDCSAFVCSNYAACHDLLSEYGTFCDNINSILNEVNDLLDITQLQVANALMQGVTLSSNLNTNLHSDVDNIDFKSLLGCLGSQCGSSSRSLLEDLLFNKVKLSDVGFVEAYNNCTGGSEIRDLLCVQSFNGIKVLPPILSETQISGYTTAATVAAMFPPWSAAAGVPFSLNVQYRINGLGVTMDVLNKNQKLIANAFNKALLSIQNGFTATNSALAKIQSVVNANAQALNSLLQQLFNKFGAISSSLQEILSRLDPPEAQVQIDRLINGRLTALNAYVSQQLSDITLIKAGASRAIEKVNECVKSQSPRINFCGNGNHILSLVQNAPYGLLFIHFSYKPTSFKTVLVSPGLCLSGDRGIAPKQGYFIKQNDSWMFTGSSYYYPEPISDKNVVFMNSCSVNFTKAPFIYLNNSIPNLSDFEAELSLWFKNHTSIAPNLTFNSHINATFLDLYYEMNVIQESIKSLN

A C-terminal trimerization domain can be added to the protomers of theHKU1-CoV S ectodomains trimer to promote trimerization of theectodomain.

An exemplary sequence of HKU1-CoV S ectodomain including a doubleproline substitution for stabilization in the prefusion conformation,mutations to eliminate the S1/S2 cleavage site, and a T4 fibritintrimerization domain is provided as SEQ ID NO: 31:

MFLIIFILPTTLAVIGDFNCTNSFINDYNKTIPRISEDVVDVSLGLGTYYVLNRVYLNTTLLFTGYFPKSGANFRDLALKGSIYLSTLWYKPPFLSDFNNGIFSKVKNTKLYVNNTLYSEFSTIVIGSVFVNTSYTIVVQPHNGILEITACQYTMCEYPHTVCKSKGSIRNESWHIDSSEPLCLFKKNFTYNVSADWLYFHFYQERGVFYAYYADVGMPTTFLFSLYLGTILSHYYVMPLTCNAISSNTDNETLEYWVTPLSRRQYLLNFDEHGVITNAVDCSSSFLSEIQCKTQSFAPNTGVYDLSGFTVKPVATVYRRIPNLPDCDIDNWLNNVSVPSPLNWERRIFSNCNFNLSTLLRLVHVDSFSCNNLDKSKIFGSCFNSITVDKFAIPNRRRDDLQLGSSGFLQSSNYKIDISSSSCQLYYSLPLVNVTINNFNPSSWNRRYGFGSFNLSSYDVVYSDHCFSVNSDFCPCADPSVVNSCAKSKPPSAICPAGTKYRHCDLDTTLYVKNWCRCSCLPDPISTYSPNTCPQKKVVVGIGEHCPGLGINEEKCGTQLNHSSCFCSPDAFLGWSFDSCISNNRCNIFSNFIFNGINSGTTCSNDLLYSNTEISTGVCVNYDLYGITGQGIFKEVSAAYYNNWQNLLYDSNGNIIGFKDFLTNKTYTILPCYSGRVSAAFYQNSSSPALLYRNLKCSYVLNNISFISQPFYFDSYLGCVLNAVNLTSYSVSSCDLRMGSGFCIDYALPSSGGSGSGISSPYRFVTFEPFNVSFVNDSVETVGGLFEIQIPTNFTIAGHEEFIQTSSPKVTIDCSAFVCSNYAACHDLLSEYGTFCDNINSILNEVNDLLDITQLQVANALMQGVTLSSNLNTNLHSDVDNIDFKSLLGCLGSQCGSSSRSLLEDLLFNKVKLSDVGFVEAYNNCTGGSEIRDLLCVQSFNGIKVLPPILSETQISGYTTAATVAAMFPPWSAAAGVPFSLNVQYRINGLGVTMDVLNKNQKLIANAFNKALLSIQNGFTATNSALAKIQSVVNANAQALNSLLQQLFNKFGAISSSLQEILSRLDPPEAQVQIDRLINGRLTALNAYVSQQLSDITLIKAGASRAIEKVNECVKSQSPRINFCGNGNHILSLVQNAPYGLLFIHFSYKPTSFKTVLVSPGLCLSGDRGIAPKQGYFIKQNDSWMFTGSSYYYPEPISDKNVVFMNSCSVNFTKAPFIYLNNSIPNLSDFEAELSLWFKNHTSIAPNLTFNSHINATFLDLYYEMNVIQESIKSLNGGYIPEAPRDGQAYVRKDGEWVLL STF

In some embodiments, the recombinant HKU1-CoV S ectodomain trimercomprises protomers comprising the ectodomain sequence of SEQ ID NO: 9.In some embodiments, the recombinant HKU1-CoV S ectodomain trimercomprises protomers comprising residues 14-1276 of SEQ ID NO: 9 orresidues 14-1303 of SEQ ID NO: 31. In some embodiments, the recombinantHKU1-CoV S ectodomain trimer comprises protomers comprising anectodomain sequence at least 90% identical to the ectodomain sequence ofSEQ ID NO: 9, wherein the HKU1-CoV S ectodomain trimer is stabilized inthe prefusion conformation and comprises the “2P” substitution and/ormodifications to remove the S1/S2 cleavage site and the S2′ cleavagesite of the protomers. In some embodiments, the recombinant HKU1-CoV Sectodomain trimer comprises protomers comprising an amino acid sequenceat least 90% identical to residues 14-1276 of SEQ ID NO: 9 or residues14-1303 of SEQ ID NO: 31, wherein the HKU1-CoV S ectodomain trimer isstabilized in the prefusion conformation and comprises the “2P”substitution and/or modifications to remove the S1/S2 cleavage site andthe S2′ cleavage site of the protomers.

D. HKU9-CoV

In some embodiments, the immunogen comprises a recombinant HKU9-CoV Sectodomain trimer comprising protomers comprising one or more (such astwo, for example two consecutive) proline substitutions at or near theboundary between a HR1 domain and a central helix domain that stabilizethe S ectodomain trimer in the prefusion conformation. In some suchembodiments, the one or more (such as two, for example two consecutive)proline substitutions that stabilize the S ectodomain in the prefusionconformation are located between a position 15 amino acids N-terminal ofa C-terminal residue of the HR1 and a position 5 amino acids C-terminalof a N-terminal residue of the central helix.

In some embodiments, the one or more (such as two, for example twoconsecutive) proline substitutions that stabilize the HKU9-CoV Sectodomain trimer in the prefusion conformation are located betweenresidues 966 to 986 (such as between residues 976 to 986 or betweenresidues 981 to 986) of the S ectodomain protomers in the trimer. Insome embodiments, the HKU9-CoV S ectodomain trimer is stabilized in theprefusion conformation by G1018P and L1019P substitutions (“2P”) in theS ectodomain protomers in the trimer. The amino acid numbering forHKU9-CoV S proteins is with reference to the HKU9-CoV S sequenceprovided as SEQ ID NO: 12.

In some embodiments, the recombinant HKU9-CoV S ectodomain trimerstabilized in the prefusion conformation comprises single-chain Sectodomain protomers comprising mutations to the S1/S2 and/or S2′protease cleavage sites to prevent protease cleavage at these sites.

In some embodiments, the protomers of the recombinant HKU9-CoV Sectodomain trimer stabilized in the prefusion conformation by the one ormore proline substitutions (such as G1018P and L1019P substitutions)comprises additional modifications for stabilization in the prefusionconformation.

With reference to the HKU9-CoV S protein sequence provided as SEQ ID NO:12, the ectodomain of the HKU9-CoV S protein includes about residues15-1207. Residues 1-14 are the signal peptide, which is removed duringcellular processing. The S1/S2 cleavage site is located at aboutposition 676/677. The S2′ cleavage site is located at about position809/810. The HR1 is located at about residues 912-980. The central helixis located at about residues 986-1026. The HR2 is located at about1162-1193. The C-terminal end of the S2 ectodomain is located at aboutresidue 1207. In some embodiments, the protomers of theprefusion-stabilized HKU9-CoV S ectodomain trimer can have a C-terminalresidue (which can be linked to a trimerization domain, or atransmembrane domain, for example) of the C-terminal residue of the HR2(e.g., position 1193), or the ectodomain (e.g., position 1207), or fromone of positions 1193-1207. The position numbering of the S protein mayvary between HKU9-CoV stains, but the sequences can be aligned todetermine relevant structural domains and cleavage sites. It will beappreciated that a few residues (such as up to 10) on the N andC-terminal ends of the ectodomain can be removed or modified in thedisclosed immunogens without decreasing the utility of the S ectodomaintrimer as an immunogen.

Exemplary HKU9-CoV S protein sequences are provided below. The prefusionstabilizing substitutions disclosed herein (and other modifications,such as substitutions to generate a single chain) can be incorporatedinto HKU9-CoV S protein sequences.

An exemplary sequence of HKU9-CoV S protein (including the ectodomainand TM and CT domains) is provided as SEQ ID NO: 12 (GenBankGI:148841195, incorporated by reference herein):

MLLILVLGVSLAAASRPECFNPRFTLTPLNHTLNYTSIKAKVSNVLLPDPYIAYSGQTLRQNLFMADMSNTILYPVTPPANGANGGFIYNTSIIPVSAGLFVNTWMYRQPASSRAYCQEPFGVAFGDTFENDRIAILIMAPDNLGSWSAVAPRNQTNIYLLVCSNATLCINPGFNRWGPAGSFIAPDALVDHSNSCFVNNTFSVNISTSRISLAFLFKDGDLLIYHSGWLPTSNFEHGFSRGSHPMTYFMSLPVGGNLPRAQFFQSIVRSNAIDKGDGMCTNFDVNLHVAHLINRDLLVSYFNNGSVANAADCADSAAEELYCVTGSFDPPTGVYPLSRYRAQVAGFVRVTQRGSYCTPPYSVLQDPPQPVVWRRYMLYDCVFDFTVVVDSLPTHQLQCYGVSPRRLASMCYGSVTLDVMRINETHLNNLFNRVPDTFSLYNYALPDNFYGCLHAFYLNSTAPYAVANRFPIKPGGRQSNSAFIDTVINAAHYSPFSYVYGLAVITLKPAAGSKLVCPVANDTVVITDRCVQYNLYGYTGTGVLSKNTSLVIPDGKVFTASSTGTIIGVSINSTTYSIMPCVTVPVSVGYHPNFERALLFNGLSCSQRSRAVTEPVSVLWSASATAQDAFDTPSGCVVNVELRNTTIVNTCAMPIGNSLCFINGSIATANADSLPRLQLVNYDPLYDNSTATPMTPVYWVKVPTNFTLSATEEYIQTTAPKITIDCARYLCGDSSRCLNVLLHYGTFCNDINKALSRVSTILDSALLSLVKELSINTRDEVTTFSFDGDYNFTGLMGCLGPNCGATTYRSAFSDLLYDKVRITDPGFMQSYQKCIDSQWGGSIRDLLCTQTYNGIAVLPPIVSPAMQALYTSLLVGAVASSGYTFGITSAGVIPFATQLQFRLNGIGVTTQVLVENQKLIASSFNNALVNIQKGFTETSIALSKMQDVINQHAAQLHTLVVQLGNSFGAISSSINEIFSRLEGLAANAEVDRLINGRMMVLNTYVTQLLIQASEAKAQNALAAQKISECVKAQSLRNDFCGNGTHVLSIPQLAPNGVLFIHYAYTPTEYAFVQTSAGLCHNGTGYAPRQGMFVLPNNTNMWHFTTMQFYNPVNISASNTQVLTSCSVNYTSVNYTVLEPSVPGDYDFQKEFDKFYKNLSTIFNNTFNPNDFNFSTVDVTAQIKSLHDVVNQLNQSFIDLKKLNVYEKTIKWPWYVWLAMIAGIVGLVLAVIMLMCMTNCCSCFKGMCDCRRCCGSYDSYDDVYPAVRVNKKRTV

An exemplary sequence of HKU9-CoV S protein including a double prolinesubstitution for stabilization in the prefusion conformation is providedas SEQ ID NO: 13:

MLLILVLGVSLAAASRPECFNPRFTLTPLNHTLNYTSIKAKVSNVLLPDPYIAYSGQTLRQNLFMADMSNTILYPVTPPANGANGGFIYNTSIIPVSAGLFVNTWMYRQPASSRAYCQEPFGVAFGDTFENDRIAILIMAPDNLGSWSAVAPRNQTNIYLLVCSNATLCINPGFNRWGPAGSFIAPDALVDHSNSCFVNNTFSVNISTSRISLAFLFKDGDLLIYHSGWLPTSNFEHGFSRGSHPMTYFMSLPVGGNLPRAQFFQSIVRSNAIDKGDGMCTNFDVNLHVAHLINRDLLVSYFNNGSVANAADCADSAAEELYCVTGSFDPPTGVYPLSRYRAQVAGFVRVTQRGSYCTPPYSVLQDPPQPVVWRRYMLYDCVFDFTVVVDSLPTHQLQCYGVSPRRLASMCYGSVTLDVMRINETHLNNLFNRVPDTFSLYNYALPDNFYGCLHAFYLNSTAPYAVANRFPIKPGGRQSNSAFIDTVINAAHYSPFSYVYGLAVITLKPAAGSKLVCPVANDTVVITDRCVQYNLYGYTGTGVLSKNTSLVIPDGKVFTASSTGTIIGVSINSTTYSIMPCVTVPVSVGYHPNFERALLFNGLSCSQRSRAVTEPVSVLWSASATAQDAFDTPSGCVVNVELRNTTIVNTCAMPIGNSLCFINGSIATANADSLPRLQLVNYDPLYDNSTATPMTPVYWVKVPTNFTLSATEEYIQTTAPKITIDCARYLCGDSSRCLNVLLHYGTFCNDINKALSRVSTILDSALLSLVKELSINTRDEVTTFSFDGDYNFTGLMGCLGPNCGATTYRSAFSDLLYDKVRITDPGFMQSYQKCIDSQWGGSIRDLLCTQTYNGIAVLPPIVSPAMQALYTSLLVGAVASSGYTFGITSAGVIPFATQLQFRLNGIGVTTQVLVENQKLIASSFNNALVNIQKGFTETSIALSKMQDVINQHAAQLHTLVVQLGNSFGAISSSINEIFSRLEPPAANAEVDRLINGRMMVLNTYVTQLLIQASEAKAQNALAAQKISECVKAQSLRNDFCGNGTHVLSIPQLAPNGVLFIHYAYTPTEYAFVQTSAGLCHNGTGYAPRQGMFVLPNNTNMWHFTTMQFYNPVNISASNTQVLTSCSVNYTSVNYTVLEPSVPGDYDFQKEFDKFYKNLSTIFNNTFNPNDFNFSTVDVTAQIKSLHDVVNQLNQSFIDLK KLNVYEK

A C-terminal trimerization domain can be added to the protomers of theHKU9-CoV S ectodomains trimer to promote trimerization of theectodomain.

An exemplary sequence of HKU9-CoV S protein including a double prolinesubstitution for stabilization in the prefusion conformation, and a T4fibritin trimerization domain is provided as SEQ ID NO: 32:

MLLILVLGVSLAAASRPECFNPRFTLTPLNHTLNYTSIKAKVSNVLLPDPYIAYSGQTLRQNLFMADMSNTILYPVTPPANGANGGFIYNTSIIPVSAGLFVNTWMYRQPASSRAYCQEPFGVAFGDTFENDRIAILIMAPDNLGSWSAVAPRNQTNIYLLVCSNATLCINPGFNRWGPAGSFIAPDALVDHSNSCFVNNTFSVNISTSRISLAFLFKDGDLLIYHSGWLPTSNFEHGFSRGSHPMTYFMSLPVGGNLPRAQFFQSIVRSNAIDKGDGMCTNFDVNLHVAHLINRDLLVSYFNNGSVANAADCADSAAEELYCVTGSFDPPTGVYPLSRYRAQVAGFVRVTQRGSYCTPPYSVLQDPPQPVVWRRYMLYDCVFDFTVVVDSLPTHQLQCYGVSPRRLASMCYGSVTLDVMRINETHLNNLFNRVPDTFSLYNYALPDNFYGCLHAFYLNSTAPYAVANRFPIKPGGRQSNSAFIDTVINAAHYSPFSYVYGLAVITLKPAAGSKLVCPVANDTVVITDRCVQYNLYGYTGTGVLSKNTSLVIPDGKVFTASSTGTIIGVSINSTTYSIMPCVTVPVSVGYHPNFERALLFNGLSCSQRSRAVTEPVSVLWSASATAQDAFDTPSGCVVNVELRNTTIVNTCAMPIGNSLCFINGSIATANADSLPRLQLVNYDPLYDNSTATPMTPVYWVKVPTNFTLSATEEYIQTTAPKITIDCARYLCGDSSRCLNVLLHYGTFCNDINKALSRVSTILDSALLSLVKELSINTRDEVTTFSFDGDYNFTGLMGCLGPNCGATTYRSAFSDLLYDKVRITDPGFMQSYQKCIDSQWGGSIRDLLCTQTYNGIAVLPPIVSPAMQALYTSLLVGAVASSGYTFGITSAGVIPFATQLQFRLNGIGVTTQVLVENQKLIASSFNNALVNIQKGFTETSIALSKMQDVINQHAAQLHTLVVQLGNSFGAISSSINEIFSRLEPPAANAEVDRLINGRMMVLNTYVTQLLIQASEAKAQNALAAQKISECVKAQSLRNDFCGNGTHVLSIPQLAPNGVLFIHYAYTPTEYAFVQTSAGLCHNGTGYAPRQGMFVLPNNTNMWHFTTMQFYNPVNISASNTQVLTSCSVNYTSVNYTVLEPSVPGDYDFQKEFDKFYKNLSTIFNNTFNPNDFNFSTVDVTAQIKSLHDVVNQLNQSFIDLKKLNVYEKGGYIPEAPRDGQAYVRKDGEWVLLSTF

In some embodiments, the recombinant HKU9-CoV S ectodomain trimercomprises protomers comprising the ectodomain sequence of SEQ ID NO: 13.In some embodiments, the recombinant HKU9-CoV S ectodomain trimercomprises protomers comprising residues 15-1207 of SEQ ID NO: 13 orresidues 15-1234 of SEQ ID NO: 32. In some embodiments, the recombinantHKU9-CoV S ectodomain trimer comprises protomers comprising anectodomain sequence at least 90% identical to the ectodomain sequence ofSEQ ID NO: 13, wherein the HKU9-CoV S ectodomain trimer is stabilized inthe prefusion conformation and comprises the “2P” substitution and/ormodifications to remove the S1/S2 cleavage site and the S2′ cleavagesite of the protomers. In some embodiments, the recombinant HKU9-CoV Sectodomain trimer comprises protomers comprising an amino acid sequenceat least 90% identical to residues 15-1207 of SEQ ID NO: 13 or residues15-1234 of SEQ ID NO: 32, wherein the HKU9-CoV S ectodomain trimer isstabilized in the prefusion conformation and comprises the “2P”substitution and/or modifications to remove the S1/S2 cleavage site andthe S2′ cleavage site of the protomers.

E. OC43-CoV

In some embodiments, the immunogen comprises a recombinant OC43-CoV Sectodomain trimer comprising protomers comprising one or more (such astwo, for example two consecutive) proline substitutions at or near theboundary between a HR1 domain and a central helix domain that stabilizethe S ectodomain trimer in the prefusion conformation. In some suchembodiments, the one or more (such as two, for example two consecutive)proline substitutions that stabilize the S ectodomain in the prefusionconformation are located between a position 15 amino acids N-terminal ofa C-terminal residue of the HR1 and a position 5 amino acids C-terminalof a N-terminal residue of the central helix.

In some embodiments, the one or more (such as two, for example twoconsecutive) proline substitutions that stabilize the OC43-CoV Sectodomain trimer in the prefusion conformation are located betweenresidues 1062-1082 (such as between residues 1072-1082 or betweenresidues 1077-1082) of the S ectodomain protomers in the trimer. In someembodiments, the OC43-CoV S ectodomain trimer is stabilized in theprefusion conformation by A1079P and L1080P substitutions (“2P”) in theS ectodomain protomers in the trimer. The amino acid numbering forOC43-CoV S proteins is with reference to the OC43-CoV S sequenceprovided as SEQ ID NO: 10.

In some embodiments, the recombinant OC43-CoV S ectodomain trimerstabilized in the prefusion conformation comprises single-chain Sectodomain protomers comprising mutations to the S1/S2 and/or S2′protease cleavage sites to prevent protease cleavage at these sites.

In some embodiments, the protomers of the recombinant OC43-CoV Sectodomain trimer stabilized in the prefusion conformation by the one ormore proline substitutions (such as A1079P and L1080P substitutions)comprise additional modifications for stabilization in the prefusionconformation.

With reference to the OC43-CoV S protein sequence provided as SEQ ID NO:10, the ectodomain of the OC43-CoV S protein includes about residues15-1301. Residues 1-14 are the signal peptide, which is removed duringcellular processing. The S1/S2 cleavage site is located at aboutposition 767/768. The S2′ cleavage site is located at about position912/913. The HR1 is located at about residues 1008-1076. The centralhelix is located at about residues 1081-1122. The HR2 is located atabout 1257-1287. The C-terminal end of the S2 ectodomain is located atabout residue 1301. In some embodiments, the protomers of theprefusion-stabilized OC43-CoV S ectodomain trimer can have a C-terminalresidue (which can be linked to a trimerization domain, or atransmembrane domain, for example) of the C-terminal residue of the HR2(e.g., position 1287), or the ectodomain (e.g., position 1301), or fromone of positions 1287-1301. The position numbering of the S protein mayvary between OC43-CoV stains, but the sequences can be aligned todetermine relevant structural domains and cleavage sites. It will beappreciated that a few residues (such as up to 10) on the N andC-terminal ends of the ectodomain can be removed or modified in thedisclosed immunogens without decreasing the utility of the S ectodomaintrimer as an immunogen.

Exemplary OC43-CoV S protein sequences are provided below. The prefusionstabilizing substitutions disclosed herein (and other modifications,such as substitutions to generate a single chain) can be incorporatedinto OC43-CoV S protein sequences.

An exemplary sequence of OC43-CoV S protein (including the ectodomainand TM and CT domains) is provided as GenBank GI: 744516696,incorporated by reference herein. Another exemplary sequence of OC43-CoVS protein is provided as GenBank GI:549302, incorporated by referenceherein):

(SEQ ID NO: 10) MFLILLISLPTAFAVIGDLKCPLDSRTGSLNNIDTGPPSISTATVDVTNGLGTYYVLDRVYLNTTLFLNGYYPTSGSTYRNMALKGTDKLSTLWFKPPFLSDFINGIFAKVKNTKVFKDGVMYSEFPAITIGSTFVNTSYSVVVQPRTINSTQDGVNKLQGLLEVSVCQYNMCEYPHTICHPKLGNHFKELWHMDTGVVSCLYKRNFTYDVNATYLYFHFYQEGGTFYAYFTDTGVVTKFLFNVYLGMALSHYYVMPLTCISRRDIGFTLEYWVTPLTSRQYLLAFNQDGIIFNAVDCMSDFMSEIKCKTQSIAPPTGVYELNGYTVQPIADVYRRKPDLPNCNIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADSFTCNNIDAAKIYGMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQLYYNLPAANVSVSRFNPSTWNKRFGFIENSVFKPQPAGVLTNHDVVYAQHCFKAPKNFCPCKLNSSLCVGSGPGKNNGIGTCPAGTNYLTCHNLCNPDPITFTGPYKCPQTKSLVGIGEHCSGLAVKSDYCGGNPCTCQPQAFLGWSADSCLQGDKCNIFANLILHDVNSGLTCSTDLQKANTDIKLGVCVNYDLYGISGQGIFVEVNATYYNSWQNLLYDSNGNLYGFRDYITNRTFMIRSCYSGRVSAAFHANSSEPALLFRNIKCNYVFNNSLIRQLQPINYFDSYLGCVVNAYNSTAISVQTCDLTVGSGYCVDYSKNRRSRRAITTGYRFTNFEPFTVNSVNDSLEPVGGLYEIQIPSEFTIGNMEEFIQTSSPKVTIDCAAFVCGDYAACKSQLVEYGSFCDNINAILTEVNELLDTTQLQVANSLMNGVTLSTKLKDGVNFNVDDINFSSVLGCLGSECSKASSRSAIEDLLFDKVKLSDVGFVAAYNNCTGGAEIRDLICVQSYKGIKVLPPLLSENQISGYTLAATSASLFPPWTAAAGVPFYLNVQYRINGLGVTMDVLSQNQKLIANAFNNALDAIQEGFDATNSALVKIQAVVNANAEALNNLLQQLSNRFGAISSSLQEILSRLDALEAEAQIDRLINGRLTALNAYVSQQLSDSTLVKFSAAQAMEKVNECVKSQSSRINFCGNGNHIISLVQNAPYGLYFIHFSYVPTKYVTAKVSPGLCIAGDRGIAPKSGYFVNVNNTWMYTGSGYYYPEPITENNVVVMSTCAVNYTKAPYVMLNTSTPNLPDFREELDQWFKNQTSVAPDLSLDYINVTFLDLQVEMNRLQEAIKVLNQSYINLKDIGTYEYYVKWPWYVWLLIGLAGVAMLVLLFFICCCTGCGTSCFKKCGGCCDDYTG YQELVIKTSHDD

An exemplary sequence of OC43-CoV S ectodomain including a doubleproline substitution for stabilization in the prefusion conformation isprovided as SEQ ID NO: 11, which also includes mutations to eliminatethe S1/S2 cleavage site:

MFLILLISLPTAFAVIGDLKCPLDSRTGSLNNIDTGPPSISTATVDVTNGLGTYYVLDRVYLNTTLFLNGYYPTSGSTYRNMALKGTDKLSTLWFKPPFLSDFINGIFAKVKNTKVFKDGVMYSEFPAITIGSTFVNTSYSVVVQPRTINSTQDGVNKLQGLLEVSVCQYNMCEYPHTICHPKLGNHFKELWHMDTGVVSCLYKRNFTYDVNATYLYFHFYQEGGTFYAYFTDTGVVTKFLFNVYLGMALSHYYVMPLTCISRRDIGFTLEYWVTPLTSRQYLLAFNQDGIIFNAVDCMSDFMSEIKCKTQSIAPPTGVYELNGYTVQPIADVYRRKPDLPNCNIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADSFTCNNIDAAKIYGMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQLYYNLPAANVSVSRFNPSTWNKRFGFIENSVFKPQPAGVLTNHDVVYAQHCFKAPKNFCPCKLNSSLCVGSGPGKNNGIGTCPAGTNYLTCHNLCNPDPITFTGPYKCPQTKSLVGIGEHCSGLAVKSDYCGGNPCTCQPQAFLGWSADSCLQGDKCNIFANLILHDVNSGLTCSTDLQKANTDIKLGVCVNYDLYGISGQGIFVEVNATYYNSWQNLLYDSNGNLYGFRDYITNRTFMIRSCYSGRVSAAFHANSSEPALLFRNIKCNYVFNNSLIRQLQPINYFDSYLGCVVNAYNSTAISVQTCDLTVGSGYCVDYSKNGGSGSAITTGYRFTNFEPFTVNSVNDSLEPVGGLYEIQIPSEFTIGNMEEFIQTSSPKVTIDCAAFVCGDYAACKSQLVEYGSFCDNINAILTEVNELLDTTQLQVANSLMNGVTLSTKLKDGVNFNVDDINFSSVLGCLGSECSKASSRSAIEDLLFDKVKLSDVGFVAAYNNCTGGAEIRDLICVQSYKGIKVLPPLLSENQISGYTLAATSASLFPPWTAAAGVPFYLNVQYRINGLGVTMDVLSQNQKLIANAFNNALDAIQEGFDATNSALVKIQAVVNANAEALNNLLQQLSNRFGAISSSLQEILSRLDPPEAEAQIDRLINGRLTALNAYVSQQLSDSTLVKFSAAQAMEKVNECVKSQSSRINFCGNGNHIISLVQNAPYGLYFIHFSYVPTKYVTAKVSPGLCIAGDRGIAPKSGYFVNVNNTWMYTGSGYYYPEPITENNVVVMSTCAVNYTKAPYVMLNTSTPNLPDFREELDQWFKNQTSVAPDLSLDYINVTFLDLQVEMNRLQEAIKVLN

A C-terminal trimerization domain can be added to the protomers of theOC43-CoV S ectodomains trimer to promote trimerization of theectodomain.

An exemplary sequence of OC43-CoV S ectodomain including a doubleproline substitution for stabilization in the prefusion conformation,mutations to eliminate the S1/S2 cleavage site, and a T4 fibritintrimerization domain is provided as SEQ ID NO: 33:

MFLILLISLPTAFAVIGDLKCPLDSRTGSLNNIDTGPPSISTATVDVTNGLGTYYVLDRVYLNTTLFLNGYYPTSGSTYRNMALKGTDKLSTLWFKPPFLSDFINGIFAKVKNTKVFKDGVMYSEFPAITIGSTFVNTSYSVVVQPRTINSTQDGVNKLQGLLEVSVCQYNMCEYPHTICHPKLGNHFKELWHMDTGVVSCLYKRNFTYDVNATYLYFHFYQEGGTFYAYFTDTGVVTKFLFNVYLGMALSHYYVMPLTCISRRDIGFTLEYWVTPLTSRQYLLAFNQDGIIFNAVDCMSDFMSEIKCKTQSIAPPTGVYELNGYTVQPIADVYRRKPDLPNCNIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADSFTCNNIDAAKIYGMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQLYYNLPAANVSVSRFNPSTWNKRFGFIENSVFKPQPAGVLTNHDVVYAQHCFKAPKNFCPCKLNSSLCVGSGPGKNNGIGTCPAGTNYLTCHNLCNPDPITFTGPYKCPQTKSLVGIGEHCSGLAVKSDYCGGNPCTCQPQAFLGWSADSCLQGDKCNIFANLILHDVNSGLTCSTDLQKANTDIKLGVCVNYDLYGISGQGIFVEVNATYYNSWQNLLYDSNGNLYGFRDYITNRTFMIRSCYSGRVSAAFHANSSEPALLFRNIKCNYVFNNSLIRQLQPINYFDSYLGCVVNAYNSTAISVQTCDLTVGSGYCVDYSKNGGSGSAITTGYRFTNFEPFTVNSVNDSLEPVGGLYEIQIPSEFTIGNMEEFIQTSSPKVTIDCAAFVCGDYAACKSQLVEYGSFCDNINAILTEVNELLDTTQLQVANSLMNGVTLSTKLKDGVNFNVDDINFSSVLGCLGSECSKASSRSAIEDLLFDKVKLSDVGFVAAYNNCTGGAEIRDLICVQSYKGIKVLPPLLSENQISGYTLAATSASLFPPWTAAAGVPFYLNVQYRINGLGVTMDVLSQNQKLIANAFNNALDAIQEGFDATNSALVKIQAVVNANAEALNNLLQQLSNRFGAISSSLQEILSRLDPPEAEAQIDRLINGRLTALNAYVSQQLSDSTLVKFSAAQAMEKVNECVKSQSSRINFCGNGNHIISLVQNAPYGLYFIHFSYVPTKYVTAKVSPGLCIAGDRGIAPKSGYFVNVNNTWMYTGSGYYYPEPITENNVVVMSTCAVNYTKAPYVMLNTSTPNLPDFREELDQWFKNQTSVAPDLSLDYINVTFLDLQVEMNRLQEAIKVLNGGYIPEAPRDGQA YVRKDGEWVLLSTF

In some embodiments, the recombinant OC43-CoV S ectodomain trimercomprises protomers comprising the ectodomain sequence of SEQ ID NO: 11.In some embodiments, the recombinant OC43-CoV S ectodomain trimercomprises protomers comprising residues 15-1287 of SEQ ID NO: 11 orresidues 15-1314 of SEQ ID NO: 33. In some embodiments, the recombinantOC43-CoV S ectodomain trimer comprises protomers comprising anectodomain sequence at least 90% identical to the ectodomain sequence ofSEQ ID NO: 11 or residues 15-1314 of SEQ ID NO: 33, wherein the OC43-CoVS ectodomain trimer is stabilized in the prefusion conformation andcomprises the “2P” substitution and/or modifications to remove the S1/S2cleavage site and the S2′ cleavage site of the protomers. In someembodiments, the recombinant OC43-CoV S ectodomain trimer comprisesprotomers comprising an amino acid sequence at least 90% identical toresidues 15-1287 of SEQ ID NO: 11, wherein the OC43-CoV S ectodomaintrimer is stabilized in the prefusion conformation and comprises the“2P” substitution and/or modifications to remove the S1/S2 cleavage siteand the S2′ cleavage site of the protomers.

F. WIV1-CoV

In some embodiments, the immunogen comprises a recombinant WIV1-CoV Sectodomain trimer comprising protomers comprising one or more (such astwo, for example two consecutive) proline substitutions at or near theboundary between a HR1 domain and a central helix domain that stabilizethe S ectodomain trimer in the prefusion conformation. In some suchembodiments, the one or more (such as two, for example two consecutive)proline substitutions that stabilize the S ectodomain in the prefusionconformation are located between a position 15 amino acids N-terminal ofa C-terminal residue of the HR1 and a position 5 amino acids C-terminalof a N-terminal residue of the central helix.

In some embodiments, the one or more (such as two, for example twoconsecutive) proline substitutions that stabilize the WIV1-CoV Sectodomain trimer in the prefusion conformation are located betweenresidues 952 to 972 (such as between residues 962 to 972 or betweenresidues 967 to 972) of the S ectodomain protomers in the trimer. Insome embodiments, the WIV1-CoV S ectodomain trimer is stabilized in theprefusion conformation by K969P and V970P substitutions (“2P”) in the Sectodomain protomers in the trimer. The amino acid numbering forWIV1-CoV S proteins is with reference to the WIV1-CoV S sequenceprovided as SEQ ID NO: 14.

In some embodiments, the recombinant WIV1-CoV S ectodomain trimerstabilized in the prefusion conformation comprises single-chain Sectodomain protomers comprising mutations to the S1/S2 and/or S2′protease cleavage sites to prevent protease cleavage at these sites.

In some embodiments, the protomers of the recombinant WIV1-CoV Sectodomain trimer stabilized in the prefusion conformation by the one ormore proline substitutions (such as K969P and V970P substitutions)comprises additional modifications for stabilization in the prefusionconformation.

With reference to the WIV1-CoV S protein sequence provided as SEQ ID NO:14, the ectodomain of the WIV1-CoV S protein includes about residues16-1191. Residues 1-15 are the signal peptide, which is removed duringcellular processing. The S1/S2 cleavage site is located at aboutposition 668/669. The S2′ cleavage site is located at about position798/799. The HR1 is located at about residues 898-996. The central helixis located at about residues 971-1012. The HR2 is located at about1146-1177. The C-terminal end of the S2 ectodomain is located at aboutresidue 1191. In some embodiments, the protomers of theprefusion-stabilized WIV1-CoV S ectodomain trimer can have a C-terminalresidue (which can be linked to a trimerization domain, or atransmembrane domain, for example) of the C-terminal residue of the HR2(e.g., position 1177), or the ectodomain (e.g., position 1191), or fromone of positions 1177-1191. The position numbering of the S protein mayvary between WIV1-CoV stains, but the sequences can be aligned todetermine relevant structural domains and cleavage sites. It will beappreciated that a few residues (such as up to 10) on the N andC-terminal ends of the ectodomain can be removed or modified in thedisclosed immunogens without decreasing the utility of the S ectodomaintrimer as an immunogen.

Exemplary WIV1-CoV S protein sequences are provided below. The prefusionstabilizing substitutions disclosed herein (and other modifications,such as substitutions to generate a single chain) can be incorporatedinto WIV1-CoV S protein sequences.

An exemplary sequence of WIV1-CoV S protein (including the ectodomainand TM and CT domains) is provided as SEQ ID NO: 14 (GenBank GI:556015140, incorporated by reference herein):

MKLLVLVFATLVSSYTIEKCLDFDDRTPPANTQFLSSHRGVYYPDDIFRSNVLHLVQDHFLPFDSNVTRFITFGLNFDNPIIPFKDGIYFAATEKSNVIRGWVFGSTMNNKSQSVIIMNNSTNLVIRACNFELCDNPFFVVLKSNNTQIPSYIFNNAFNCTFEYVSKDFNLDLGEKPGNFKDLREFVFRNKDGFLHVYSGYQPISAASGLPTGFNALKPIFKLPLGINITNFRTLLTAFPPRPDYWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVAPSKEVVRFPNITNLCPFGEVFNATTFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFTGCVLAWNTRNIDATQTGNYNYKYRSLRHGKLRPFERDISNVPFSPDGKPCTPPAFNCYWPLNDYGFYITNGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNTSSEVAVLYQDVNCTDVPVAIHADQLTPSWRVHSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSSLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAVEQDRNTREVFAQVKQMYKTPTLKDFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGSCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKG VKLHYT

An exemplary sequence of WIV1-CoV S protein including a double prolinesubstitution for stabilization in the prefusion conformation is providedas SEQ ID NO: 15:

MKLLVLVFATLVSSYTIEKCLDFDDRTPPANTQFLSSHRGVYYPDDIFRSNVLHLVQDHFLPFDSNVTRFITFGLNFDNPIIPFKDGIYFAATEKSNVIRGWVFGSTMNNKSQSVIIMNNSTNLVIRACNFELCDNPFFVVLKSNNTQIPSYIFNNAFNCTFEYVSKDFNLDLGEKPGNFKDLREFVFRNKDGFLHVYSGYQPISAASGLPTGFNALKPIFKLPLGINITNFRTLLTAFPPRPDYWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVAPSKEVVRFPNITNLCPFGEVFNATTFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFTGCVLAWNTRNIDATQTGNYNYKYRSLRHGKLRPFERDISNVPFSPDGKPCTPPAFNCYWPLNDYGFYITNGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNTSSEVAVLYQDVNCTDVPVAIHADQLTPSWRVHSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSSLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAVEQDRNTREVFAQVKQMYKTPTLKDFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGSCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

A C-terminal trimerization domain can be added to the protomers of theWIV1-CoV S ectodomains trimer to promote trimerization of theectodomain.

An exemplary sequence of WIV1-CoV S protein including a double prolinesubstitution for stabilization in the prefusion conformation, and a T4fibritin trimerization domain is provided as SEQ ID NO: 34:

MKLLVLVFATLVSSYTIEKCLDFDDRTPPANTQFLSSHRGVYYPDDIFRSNVLHLVQDHFLPFDSNVTRFITFGLNFDNPIIPFKDGIYFAATEKSNVIRGWVFGSTMNNKSQSVIIMNNSTNLVIRACNFELCDNPFFVVLKSNNTQIPSYIFNNAFNCTFEYVSKDFNLDLGEKPGNFKDLREFVFRNKDGFLHVYSGYQPISAASGLPTGFNALKPIFKLPLGINITNFRTLLTAFPPRPDYWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVAPSKEVVRFPNITNLCPFGEVFNATTFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFTGCVLAWNTRNIDATQTGNYNYKYRSLRHGKLRPFERDISNVPFSPDGKPCTPPAFNCYWPLNDYGFYITNGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNTSSEVAVLYQDVNCTDVPVAIHADQLTPSWRVHSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSSLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAVEQDRNTREVFAQVKQMYKTPTLKDFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGSCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGGYIPEAPR DGQAYVRKDGEWVLLSTF

In some embodiments, the recombinant WIV1-CoV S ectodomain trimercomprises protomers comprising the ectodomain sequence of SEQ ID NO: 15.In some embodiments, the recombinant WIV1-CoV S ectodomain trimercomprises protomers comprising residues 16-1191 of SEQ ID NO: 15 orresidues 16-1218 of SEQ ID NO: 34. In some embodiments, the recombinantWIV1-CoV S ectodomain trimer comprises protomers comprising anectodomain sequence at least 90% identical to the ectodomain sequence ofSEQ ID NO: 15, wherein the WIV1-CoV S ectodomain trimer is stabilized inthe prefusion conformation and comprises the “2P” substitution and/ormodifications to remove the S1/S2 cleavage site and the S2′ cleavagesite of the protomers. In some embodiments, the recombinant WIV1-CoV Sectodomain trimer comprises protomers comprising an amino acid sequenceat least 90% identical to residues 16-1191 of SEQ ID NO: 15 or residues16-1218 of SEQ ID NO: 34, wherein the WIV1-CoV S ectodomain trimer isstabilized in the prefusion conformation and comprises the “2P”substitution and/or modifications to remove the S1/S2 cleavage site andthe S2′ cleavage site of the protomers.

G. MHV-CoV

In some embodiments, the immunogen comprises a recombinant MHV-CoV Sectodomain trimer comprising protomers comprising one or more (such astwo, for example two consecutive) proline substitutions at or near theboundary between a HR1 domain and a central helix domain that stabilizethe S ectodomain trimer in the prefusion conformation. In some suchembodiments, the one or more (such as two, for example two consecutive)proline substitutions that stabilize the S ectodomain in the prefusionconformation are located between a position 15 amino acids N-terminal ofa C-terminal residue of the HR1 and a position 5 amino acids C-terminalof a N-terminal residue of the central helix.

In some embodiments, the one or more (such as two, for example twoconsecutive) proline substitutions that stabilize the MHV-CoV Sectodomain trimer in the prefusion conformation are located betweenresidues 852 to 872 (such as between residues 862 to 872 or betweenresidues 867 to 872) of the S ectodomain protomers in the trimer. Insome embodiments, the MHV-CoV S ectodomain trimer is stabilized in theprefusion conformation by I869P and I870P substitutions (“2P”) in the Sectodomain protomers in the trimer. The amino acid numbering for MHV-CoVS proteins is with reference to the MHV-CoV S sequence provided as SEQID NO: 16.

In some embodiments, the recombinant MHV-CoV S ectodomain trimerstabilized in the prefusion conformation comprises single-chain Sectodomain protomers comprising mutations to the S1/S2 and/or S2′protease cleavage sites to prevent protease cleavage at these sites.

In some embodiments, the protomers of the recombinant MHV-CoV Sectodomain trimer stabilized in the prefusion conformation by the one ormore proline substitutions (such as I869P and 1870P substitutions)comprises additional modifications for stabilization in the prefusionconformation.

With reference to the MHV-CoV S protein sequence provided as SEQ ID NO:16, the ectodomain of the MHV-CoV S protein includes about residues15-1297. Residues 1-14 are the signal peptide, which is removed duringcellular processing. The S1/S2 cleavage site is located at aboutposition 757/758. The S2′ cleavage site is located at about position906/907. The HR1 is located at about residues 1002-1070. The centralhelix is located at about residues 1075-1116. The HR2 is located atabout 1252-1283. The C-terminal end of the S2 ectodomain is located atabout residue 1297. In some embodiments, the protomers of theprefusion-stabilized MHV-CoV S ectodomain trimer can have a C-terminalresidue (which can be linked to a trimerization domain, or atransmembrane domain, for example) of the C-terminal residue of the HR2(e.g., position 1283), or the ectodomain (e.g., position 1297), or fromone of positions 1283-1297. The position numbering of the S protein mayvary between MHV-CoV stains, but the sequences can be aligned todetermine relevant structural domains and cleavage sites. It will beappreciated that a few residues (such as up to 10) on the N andC-terminal ends of the ectodomain can be removed or modified in thedisclosed immunogens without decreasing the utility of the S ectodomaintrimer as an immunogen.

Exemplary MHV-CoV S protein sequences are provided below. The prefusionstabilizing substitutions disclosed herein (and other modifications,such as substitutions to generate a single chain) can be incorporatedinto MHV-CoV S protein sequences.

An exemplary sequence of MHV-CoV S protein (including the ectodomain andTM and CT domains) is provided as SEQ ID NO: 16 (GenBank GI:328496819,incorporated by reference herein). Another exemplary MHV-CoV sequence isprovided as GenBank GI:81971726, incorporated by reference herein:

MLSVFILFLPSCLGYIGDFRCINLVNTDTSNASAPSVSTEVVDVSKGIGTYYVLDRVYLNATLLLTGYYPVDGSNYRNLALTGTNTLSLNWYKPPFLSEFNDGIFAKVKNLKASLPKDSTSYFPTIVIGSNFVTTSYTVVLEPYNGIIMASICQYTICLLPYTDCKPNTGGNKLIGFWHIDLKSPVCILKRNFTFNVNADWLYFHFYQQGGTFYAYYADAGSATTFLFSSYIGDVLTQYFVLPFVCTPTTTGVFSPQYWVTPLVKRQYLFNFNQKGTITSAVDCASSYTSEIKCKTQSMNPNTGVYDLSGYTVQPVGLVYRRVRNLPDCRIEDWLAAKTVPSPLNWERKTFQNCNFNLSSLLRLVQAGSLSCSNIDAAKVYGMCFGSMSIDKFAIPNSRRVDLQLGNSGFLQSFNYKIDTRATSCQLYYSLAQSNVTVNNHNPSSWNRRYGFNDVATFGRGKHDVAYAEACFTVGASYCPCANPSIVSPCTTGKPKFANCPTGTTNRECNVLALGSNLFKCDCTCNPSPLTTYDLRCLQGRSMLGVGDHCEGLGVLEDKCGGSNTCNCSADAFVGWAKDSCLSNGRCHIFSNLMLNGINSGTTCSTDLQLPNTEVVTGICVKYDLYGITGQGVFKEVKADYYNSWQNLLYDVNGNLNGFRDIVTNKTYLTRSCYSGRVSAAYHQDAPEPALLYRNLKCDYVFNNNIFREETPLNYFDSYLGCVVNADNSTEQAVDACDLRMGSGLCVNYSTAHRARTSVSTGYKLTTFEPFTVSIVNDSVESVGGLYEMQIPTNFTIASHQEFIQTRAPKVTIDCAAFVCGDYTTCRQQLVEYGSFCDNINAILGEVNNLIDTMQLQVASALIQGVTLSSRLADGISGQIDDINFSPLLGCLGSQCSEGTMAAQGRSTVEDLLFDKVKLSDVGFVEAYNNCTGGQEVRDLLCVQSFNGIKVLPPVLSENQVSGYTAGATASSMFPPWSAAAGVPFSLSVQYRINGLGVTMNVLSENQKMIASAFNNAIGAIQEGFDATNSALAKIQSVVNANAEALNNLLNQLSNRFGAISASLQEILSRLDALEAQAQIDRLINGRLTALNAYVSKQLSDMTLIKVSAAQAIEKVNECVKSQSPRINFCGNGNHILSLVQNAPYGLYFLHFSYVPTSFTTANVSPGLCISGDRGLAPKAGYFVQDDGEWKFTGSNYYYPEPITDKNSVVMSSCAVNYTKAPEVFLNTSISNLPDFKEELDKWFKNQTSVAPDLSLDFEKLNVTFLDLSDEMNRIQEAIKKLNESYINLKEIGTYEMYVKWPWYVWLLIGLAGVAVCVLLFFICCCTGCGSCCFKKCGNCCDEYGGHQDS IVIHNISSHED

An exemplary sequence of MHV-CoV S ectodomain including a double prolinesubstitution for stabilization in the prefusion conformation is providedas SEQ ID NO: 17:

MLSVFILFLPSCLGYIGDFRCINLVNTDTSNASAPSVSTEVVDVSKGIGTYYVLDRVYLNATLLLTGYYPVDGSNYRNLALTGTNTLSLNWYKPPFLSEFNDGIFAKVKNLKASLPKDSTSYFPTIVIGSNFVTTSYTVVLEPYNGIIMASICQYTICLLPYTDCKPNTGGNKLIGFWHIDLKSPVCILKRNFTFNVNADWLYFHFYQQGGTFYAYYADAGSATTFLFSSYIGDVLTQYFVLPFVCTPTTTGVFSPQYWVTPLVKRQYLFNFNQKGTITSAVDCASSYTSEIKCKTQSMNPNTGVYDLSGYTVQPVGLVYRRVRNLPDCRIEDWLAAKTVPSPLNWERKTFQNCNFNLSSLLRLVQAGSLSCSNIDAAKVYGMCFGSMSIDKFAIPNSRRVDLQLGNSGFLQSFNYKIDTRATSCQLYYSLAQSNVTVNNHNPSSWNRRYGFNDVATFGRGKHDVAYAEACFTVGASYCPCANPSIVSPCTTGKPKFANCPTGTTNRECNVLALGSNLFKCDCTCNPSPLTTYDLRCLQGRSMLGVGDHCEGLGVLEDKCGGSNTCNCSADAFVGWAKDSCLSNGRCHIFSNLMLNGINSGTTCSTDLQLPNTEVVTGICVKYDLYGITGQGVFKEVKADYYNSWQNLLYDVNGNLNGFRDIVTNKTYLTRSCYSGRVSAAYHQDAPEPALLYRNLKCDYVFNNNIFREETPLNYFDSYLGCVVNADNSTEQAVDACDLRMGSGLCVNYSTAHRARTSVSTGYKLTTFEPFTVSIVNDSVESVGGLYEMQIPTNFTIASHQEFIQTRAPKVTIDCAAFVCGDYTTCRQQLVEYGSFCDNINAILGEVNNLIDTMQLQVASALIQGVTLSSRLADGISGQIDDINFSPLLGCLGSQCSEGTMAAQGRSTVEDLLFDKVKLSDVGFVEAYNNCTGGQEVRDLLCVQSFNGIKVLPPVLSENQVSGYTAGATASSMFPPWSAAAGVPFSLSVQYRINGLGVTMNVLSENQKMIASAFNNAIGAIQEGFDATNSALAKIQSVVNANAEALNNLLNQLSNRFGAISASLQEILSRLDPPEAQAQIDRLINGRLTALNAYVSKQLSDMTLIKVSAAQAIEKVNECVKSQSPRINFCGNGNHILSLVQNAPYGLYFLHFSYVPTSFTTANVSPGLCISGDRGLAPKAGYFVQDDGEWKFTGSNYYYPEPITDKNSVVMSSCAVNYTKAPEVFLNTSISNLPDFKEELDKWFKNQTSVAPDLSLDFEKLNVTFLDLSDEMNRIQEAIKKLNESYINLKEIGTYEM

A C-terminal trimerization domain can be added to the protomers of theMHV-CoV S ectodomains trimer to promote trimerization of the ectodomain.

An exemplary sequence of MHV-CoV S ectodomain including a double prolinesubstitution for stabilization in the prefusion conformation, and a T4fibritin trimerization domain is provided as SEQ ID NO: 35:

MLSVFILFLPSCLGYIGDFRCINLVNTDTSNASAPSVSTEVVDVSKGIGTYYVLDRVYLNATLLLTGYYPVDGSNYRNLALTGTNTLSLNWYKPPFLSEFNDGIFAKVKNLKASLPKDSTSYFPTIVIGSNFVTTSYTVVLEPYNGIIMASICQYTICLLPYTDCKPNTGGNKLIGFWHIDLKSPVCILKRNFTFNVNADWLYFHFYQQGGTFYAYYADAGSATTFLFSSYIGDVLTQYFVLPFVCTPTTTGVFSPQYWVTPLVKRQYLFNFNQKGTITSAVDCASSYTSEIKCKTQSMNPNTGVYDLSGYTVQPVGLVYRRVRNLPDCRIEDWLAAKTVPSPLNWERKTFQNCNFNLSSLLRLVQAGSLSCSNIDAAKVYGMCFGSMSIDKFAIPNSRRVDLQLGNSGFLQSFNYKIDTRATSCQLYYSLAQSNVTVNNHNPSSWNRRYGFNDVATFGRGKHDVAYAEACFTVGASYCPCANPSIVSPCTTGKPKFANCPTGTTNRECNVLALGSNLFKCDCTCNPSPLTTYDLRCLQGRSMLGVGDHCEGLGVLEDKCGGSNTCNCSADAFVGWAKDSCLSNGRCHIFSNLMLNGINSGTTCSTDLQLPNTEVVTGICVKYDLYGITGQGVFKEVKADYYNSWQNLLYDVNGNLNGFRDIVTNKTYLTRSCYSGRVSAAYHQDAPEPALLYRNLKCDYVFNNNIFREETPLNYFDSYLGCVVNADNSTEQAVDACDLRMGSGLCVNYSTAHRARTSVSTGYKLTTFEPFTVSIVNDSVESVGGLYEMQIPTNFTIASHQEFIQTRAPKVTIDCAAFVCGDYTTCRQQLVEYGSFCDNINAILGEVNNLIDTMQLQVASALIQGVTLSSRLADGISGQIDDINFSPLLGCLGSQCSEGTMAAQGRSTVEDLLFDKVKLSDVGFVEAYNNCTGGQEVRDLLCVQSFNGIKVLPPVLSENQVSGYTAGATASSMFPPWSAAAGVPFSLSVQYRINGLGVTMNVLSENQKMIASAFNNAIGAIQEGFDATNSALAKIQSVVNANAEALNNLLNQLSNRFGAISASLQEILSRLDPPEAQAQIDRLINGRLTALNAYVSKQLSDMTLIKVSAAQAIEKVNECVKSQSPRINFCGNGNHILSLVQNAPYGLYFLHFSYVPTSFTTANVSPGLCISGDRGLAPKAGYFVQDDGEWKFTGSNYYYPEPITDKNSVVMSSCAVNYTKAPEVFLNTSISNLPDFKEELDKWFKNQTSVAPDLSLDFEKLNVTFLDLSDEMNRIQEAIKKLNESYINLKEIGTYEMGGYIPEAPRDGQAYVRKDGEWVLLSTF

In some embodiments, the recombinant MHV-CoV S ectodomain trimercomprises protomers comprising the ectodomain sequence of SEQ ID NO: 17.In some embodiments, the recombinant MHV-CoV S ectodomain trimercomprises protomers comprising residues 15-1297 of SEQ ID NO: 17 orresidues 15-1324 of SEQ ID NO: 35. In some embodiments, the recombinantMHV-CoV S ectodomain trimer comprises protomers comprising an ectodomainsequence at least 90% identical to the ectodomain sequence of SEQ ID NO:17, wherein the MHV-CoV S ectodomain trimer is stabilized in theprefusion conformation and comprises the “2P” substitution and/ormodifications to remove the S1/S2 cleavage site and the S2′ cleavagesite of the protomers. In some embodiments, the recombinant MHV-CoV Sectodomain trimer comprises protomers comprising an amino acid sequenceat least 90% identical to residues 15-1297 of SEQ ID NO: 17 or residues15-1324 of SEQ ID NO: 35, wherein the MHV-CoV S ectodomain trimer isstabilized in the prefusion conformation and comprises the “2P”substitution and/or modifications to remove the S1/S2 cleavage site andthe S2′ cleavage site of the protomers.

H. NL63-CoV

In some embodiments, the immunogen comprises a recombinant NL63-CoV Sectodomain trimer comprising protomers comprising one or more (such astwo, for example two consecutive) proline substitutions at or near theboundary between a HR1 domain and a central helix domain that stabilizethe S ectodomain trimer in the prefusion conformation. In some suchembodiments, the one or more (such as two, for example two consecutive)proline substitutions that stabilize the S ectodomain in the prefusionconformation are located between a position 15 amino acids N-terminal ofa C-terminal residue of the HR1 and a position 5 amino acids C-terminalof a N-terminal residue of the central helix.

In some embodiments, the one or more (such as two, for example twoconsecutive) proline substitutions that stabilize the NL63-CoV Sectodomain trimer in the prefusion conformation are located betweenresidues 1035 to 1055 (such as between residues 1045 to 1055 or betweenresidues 1050 to 1055) of the S ectodomain protomers in the trimer. Insome embodiments, the NL63-CoV S ectodomain trimer is stabilized in theprefusion conformation by S1052P and I1053P substitutions (“2P”) in theS ectodomain protomers in the trimer. The amino acid numbering forNL63-CoV S proteins is with reference to the NL63-CoV S sequenceprovided as SEQ ID NO: 18.

In some embodiments, the recombinant NL63-CoV S ectodomain trimerstabilized in the prefusion conformation comprises single-chain Sectodomain protomers comprising mutations to the S1/S2 and/or S2′protease cleavage sites to prevent protease cleavage at these sites.

In some embodiments, the protomers of the recombinant NL63-CoV Sectodomain trimer stabilized in the prefusion conformation by the one ormore proline substitutions (such as S1052P and I1053P substitutions)comprises additional modifications for stabilization in the prefusionconformation.

With reference to the NL63-CoV S protein sequence provided as SEQ ID NO:18, the ectodomain of the NL63-CoV S protein includes about residues16-1291. Residues 1-15 are the signal peptide, which is removed duringcellular processing. The S1/S2 cleavage site is located at aboutposition 748/749. The S2′ cleavage site is located at about position870/871. The HR1 is located at about residues 967-1049. The centralhelix is located at about residues 1054-1095. The HR2 is located atabout 1246-1272. The C-terminal end of the S2 ectodomain is located atabout residue 1291. In some embodiments, the protomers of theprefusion-stabilized NL63-CoV S ectodomain trimer can have a C-terminalresidue (which can be linked to a trimerization domain, or atransmembrane domain, for example) of the C-terminal residue of the HR2(e.g., position 1277), or the ectodomain (e.g., position 1291), or fromone of positions 1277-1291. The position numbering of the S protein mayvary between NL63-CoV stains, but the sequences can be aligned todetermine relevant structural domains and cleavage sites. It will beappreciated that a few residues (such as up to 10) on the N andC-terminal ends of the ectodomain can be removed or modified in thedisclosed immunogens without decreasing the utility of the S ectodomaintrimer as an immunogen.

Exemplary NL63-CoV S protein sequences are provided below. The prefusionstabilizing substitutions disclosed herein (and other modifications,such as substitutions to generate a single chain) can be incorporatedinto NL63-CoV S protein sequences.

An exemplary sequence of NL63-CoV S protein (including the ectodomainand TM and CT domains) is provided as SEQ ID NO: 18 (GenBank GI:71153773, incorporated by reference herein):

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWFCANQSTSVYSANGFFYIDVGNHRSAFALHTGYYDANQYYIYVTNEIGLNASVTLKICKFSRNTTFDFLSNASSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKCYFNYSCVFSVVNATVTVNVTTHNGRVVNYTVCDDCNGYTDNIFSVQQDGRIPNGFPFNNWFLLTNGSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGYQHNSVVDVMRYNLNFSANSLDNLKSGVIVFKTLQYDVLFYCSNSSSGVLDTTIPFGPSSQPYYCFINSTINTTHVSTFVGILPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFNVTTASATDFWTVAFATFVDVLVNVSATNIQNLLYCDSPFEKLQCEHLQFGLQDGFYSANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCVRTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVEVPGSCNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYDYVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIGAMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRNSSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYTSACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIRSSRIAGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADAERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASFNKAINNIVASFSSVNDAITQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQAISNSIQAIYDRLDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRGSRRLAQQKINECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGIYGYVLRQPNLVLYSDNGVFRVTSRIMFQPRLPVLSDFVQIYNCNVTFVNISRVELHTVIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTTVELQGLIDQINSTYVDLKLLNRFENYIKWPWWVWLIISVVFVVLLSLLVFCCLSTGCCGCCNCLTSSMRGCCDCGSTKLPYYEF EKVHVQ

An exemplary sequence of NL63-CoV S ectodomain including a doubleproline substitution for stabilization in the prefusion conformation isprovided as SEQ ID NO: 19:

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWFCANQSTSVYSANGFFYIDVGNHRSAFALHTGYYDANQYYIYVTNEIGLNASVTLKICKFSRNTTFDFLSNASSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKCYFNYSCVFSVVNATVTVNVTTHNGRVVNYTVCDDCNGYTDNIFSVQQDGRIPNGFPFNNWFLLTNGSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGYQHNSVVDVMRYNLNFSANSLDNLKSGVIVFKTLQYDVLFYCSNSSSGVLDTTIPFGPSSQPYYCFINSTINTTHVSTFVGILPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFNVTTASATDFWTVAFATFVDVLVNVSATNIQNLLYCDSPFEKLQCEHLQFGLQDGFYSANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCVRTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVEVPGSCNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYDYVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIGAMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRNSSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYTSACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIRSSRIAGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADAERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASFNKAINNIVASFSSVNDAITQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQAISNSIQAIYDRLDPPQADQQVDRLITGRLAALNAFVSQVLNKYTEVRGSRRLAQQKINECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGIYGYVLRQPNLVLYSDNGVFRVTSRIMFQPRLPVLSDFVQIYNCNVTFVNISRVELHTVIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTTVELQGLIDQINSTYVDLKLLNRFEN

A C-terminal trimerization domain can be added to the protomers of theNL63-CoV S ectodomains trimer to promote trimerization of theectodomain.

An exemplary sequence of NL63-CoV S ectodomain including a doubleproline substitution for stabilization in the prefusion conformation,and a T4 fibritin trimerization domain is provided as SEQ ID NO: 36:

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWFCANQSTSVYSANGFFYIDVGNHRSAFALHTGYYDANQYYIYVTNEIGLNASVTLKICKFSRNTTFDFLSNASSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKCYFNYSCVFSVVNATVTVNVTTHNGRVVNYTVCDDCNGYTDNIFSVQQDGRIPNGFPFNNWFLLTNGSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGYQHNSVVDVMRYNLNFSANSLDNLKSGVIVFKTLQYDVLFYCSNSSSGVLDTTIPFGPSSQPYYCFINSTINTTHVSTFVGILPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFNVTTASATDFWTVAFATFVDVLVNVSATNIQNLLYCDSPFEKLQCEHLQFGLQDGFYSANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCVRTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVEVPGSCNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYDYVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIGAMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRNSSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYTSACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIRSSRIAGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADAERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASFNKAINNIVASFSSVNDAITQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQAISNSIQAIYDRLDPPQADQQVDRLITGRLAALNAFVSQVLNKYTEVRGSRRLAQQKINECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGIYGYVLRQPNLVLYSDNGVFRVTSRIMFQPRLPVLSDFVQIYNCNVTFVNISRVELHTVIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTTVELQGLIDQINSTYVDLKLLNRFENGGYIPEAPR DGQAYVRKDGEWVLLSTF

In some embodiments, the recombinant NL63-CoV S ectodomain trimercomprises protomers comprising the ectodomain sequence of SEQ ID NO: 19.In some embodiments, the recombinant NL63-CoV S ectodomain trimercomprises protomers comprising residues 16-1291 of SEQ ID NO: 19 orresidues 16-1318 of SEQ ID NO: 36. In some embodiments, the recombinantNL63-CoV S ectodomain trimer comprises protomers comprising anectodomain sequence at least 90% identical to the ectodomain sequence ofSEQ ID NO: 19, wherein the NL63-CoV S ectodomain trimer is stabilized inthe prefusion conformation and comprises the “2P” substitution and/ormodifications to remove the S1/S2 cleavage site and the S2′ cleavagesite of the protomers. In some embodiments, the recombinant NL63-CoV Sectodomain trimer comprises protomers comprising an amino acid sequenceat least 90% identical to residues 16-1291 of SEQ ID NO: 19 or residues16-1318 of SEQ ID NO: 36, wherein the NL63-CoV S ectodomain trimer isstabilized in the prefusion conformation and comprises the “2P”substitution and/or modifications to remove the S1/S2 cleavage site andthe S2′ cleavage site of the protomers.

I. 229E-CoV

In some embodiments, the immunogen comprises a recombinant 229E-CoV Sectodomain trimer comprising protomers comprising one or more (such astwo, for example two consecutive) proline substitutions at or near theboundary between a HR1 domain and a central helix domain that stabilizethe S ectodomain trimer in the prefusion conformation. In some suchembodiments, the one or more (such as two, for example two consecutive)proline substitutions that stabilize the S ectodomain in the prefusionconformation are located between a position 15 amino acids N-terminal ofa C-terminal residue of the HR1 and a position 5 amino acids C-terminalof a N-terminal residue of the central helix.

In some embodiments, the one or more (such as two, for example twoconsecutive) proline substitutions that stabilize the 229E-CoV Sectodomain trimer in the prefusion conformation are located betweenresidues 852 to 872 (such as between residues 862 to 872 or betweenresidues 867 to 872) of the S ectodomain protomers in the trimer. Insome embodiments, the 229E-CoV S ectodomain trimer is stabilized in theprefusion conformation by I869P and I870P substitutions (“2P”) in the Sectodomain protomers in the trimer. The amino acid numbering for229E-CoV S proteins is with reference to the 229E-CoV S sequenceprovided as SEQ ID NO: 20.

In some embodiments, the recombinant 229E-CoV S ectodomain trimerstabilized in the prefusion conformation comprises single-chain Sectodomain protomers comprising mutations to the S1/S2 and/or S2′protease cleavage sites to prevent protease cleavage at these sites.

In some embodiments, the protomers of the recombinant 229E-CoV Sectodomain trimer stabilized in the prefusion conformation by the one ormore proline substitutions (such as I869P and 1870P substitutions)comprises additional modifications for stabilization in the prefusionconformation.

With reference to the 229E-CoV S protein sequence provided as SEQ ID NO:20, the ectodomain of the 229E-CoV S protein includes about residues17-1108. Residues 1-16 are the signal peptide, which is removed duringcellular processing. The S1/S2 cleavage site is located at aboutposition 565/566. The S2′ cleavage site is located at about position687/688. The HR1 is located at about residues 784-866. The central helixis located at about residues 871-912. The HR2 is located at about1050-1094. The C-terminal end of the S2 ectodomain is located at aboutresidue 1108. In some embodiments, the protomers of theprefusion-stabilized 229E-CoV S ectodomain trimer can have a C-terminalresidue (which can be linked to a trimerization domain, or atransmembrane domain, for example) of the C-terminal residue of the HR2(e.g., position 1099), or the ectodomain (e.g., position 1108), or fromone of positions 1099-1108. The position numbering of the S protein mayvary between 229E-CoV stains, but the sequences can be aligned todetermine relevant structural domains and cleavage sites. It will beappreciated that a few residues (such as up to 10) on the N andC-terminal ends of the ectodomain can be removed or modified in thedisclosed immunogens without decreasing the utility of the S ectodomaintrimer as an immunogen.

Exemplary 229E-CoV S protein sequences are provided below. The prefusionstabilizing substitutions disclosed herein (and other modifications,such as substitutions to generate a single chain) can be incorporatedinto 229E-CoV S protein sequences.

An exemplary sequence of 229E-CoV S protein (including the ectodomainand TM and CT domains) is provided as SEQ ID NO: 20 (GenBank GI:1060650120, incorporated by reference herein):

MFVLLVAYALLHIAGCQTTNGTNTSHSVCNGCVGHSENVFAVESGGYIPSNFAFNNWFLLTNTSSVVDGVVRSFQPLLLNCLWSVSGSQFTTGFVYFNGTGRGACKGFYSNASSDVIRYNINFEENLRRGTILFKTSYGAVVFYCTNNTLVSGDAHIPSGTVLGNFYCFVNTTIGNETTSAFVGALPKTVREFVISRTGHFYINGYRYFSLGDVEAVNFNVTNAATTVCTVALASYADVLVNVSQTAIANIIYCNSVINRLRCDQLSFDVPDGFYSTSPIQPVELPVSIVSLPVYHKHTFIVLYVNFEHRRGPGKCYNCRPAVINITLANFNETKGPLCVDTSHFTTQFVDNVKLARWSASINTGNCPFSFGKVNNFVKFGSVCFSLKDIPGGCAMPIMANLVNSKSHNIGSLYVSWSDGDVITGVPKPVEGVSSFMNVTLNKCTKYNIYDVSGVGVIRISNDTFLNGITYTSTSGNLLGFKDVTNGTIYSITPCNPPDQLVVYQQAVVGAMLSENFTSYGFSNVVEMPKFFYASNGTYNCTDAVLTYSSFGVCADGSIIAVQPRNVSYDSVSAIVTANLSIPFNWTTSVQVEYLQITSTPIVVDCSTYVCNGNVRCVELLKQYTSACKTIEDALRNSAMLESADVSEMLTFDKKAFTLANVSSFGDYNLSSVIPSLPRSGSRVAGRSAIEDILFSKLVTSGLGTVDADYKKCTKGLSIADLACAQYYNGIMVLPGVADAERMAMYTGSLIGGIALGGLTSAASIPFSLAIQSRLNYVALQTDVLQENQRILAASFNKAMTNIVDAFTGVNDAITQTSQALQTVATALNKIQDVVNQQGNSLNHLTSQLRQNFQAISSSIQAIYDRLDIIQADQQVDRLITGRLAALNVFVSHTLTKYTEVRASRQLAQQKVNECVKSQSKRYGFCGNGTHIFSLVNAAPEGLVFLHTVLLPTQYKDVEAWSGLCVDGINGYVLRQPNLALYKEGNYYRITSRIMFEPRIPTIADFVQIENCNVTFVNISRSELQTIVPEYIDVNKTLQELSYKLPNYTVPDLVVEQYNQTILNLTSEISTLENKSAELNYTVQKLQTLIDNINSTLVDLKWLNRVETYIKWPWWVWLCISVVLIFVVSMLLLCCCSTGCCGFFSCFASS IRGCCESTKLPYYDVEKIHIQ

An exemplary sequence of 229E-CoV S ectodomain including a doubleproline substitution for stabilization in the prefusion conformation isprovided as SEQ ID NO: 21:

MFVLLVAYALLHIAGCQTTNGTNTSHSVCNGCVGHSENVFAVESGGYIPSNFAFNNWFLLTNTSSVVDGVVRSFQPLLLNCLWSVSGSQFTTGFVYFNGTGRGACKGFYSNASSDVIRYNINFEENLRRGTILFKTSYGAVVFYCTNNTLVSGDAHIPSGTVLGNFYCFVNTTIGNETTSAFVGALPKTVREFVISRTGHFYINGYRYFSLGDVEAVNFNVTNAATTVCTVALASYADVLVNVSQTAIANIIYCNSVINRLRCDQLSFDVPDGFYSTSPIQPVELPVSIVSLPVYHKHTFIVLYVNFEHRRGPGKCYNCRPAVINITLANFNETKGPLCVDTSHFTTQFVDNVKLARWSASINTGNCPFSFGKVNNFVKFGSVCFSLKDIPGGCAMPIMANLVNSKSHNIGSLYVSWSDGDVITGVPKPVEGVSSFMNVTLNKCTKYNIYDVSGVGVIRISNDTFLNGITYTSTSGNLLGFKDVTNGTIYSITPCNPPDQLVVYQQAVVGAMLSENFTSYGFSNVVEMPKFFYASNGTYNCTDAVLTYSSFGVCADGSIIAVQPRNVSYDSVSAIVTANLSIPFNWTTSVQVEYLQITSTPIVVDCSTYVCNGNVRCVELLKQYTSACKTIEDALRNSAMLESADVSEMLTFDKKAFTLANVSSFGDYNLSSVIPSLPRSGSRVAGRSAIEDILFSKLVTSGLGTVDADYKKCTKGLSIADLACAQYYNGIMVLPGVADAERMAMYTGSLIGGIALGGLTSAASIPFSLAIQSRLNYVALQTDVLQENQRILAASFNKAMTNIVDAFTGVNDAITQTSQALQTVATALNKIQDVVNQQGNSLNHLTSQLRQNFQAISSSIQAIYDRLDPPQADQQVDRLITGRLAALNVFVSHTLTKYTEVRASRQLAQQKVNECVKSQSKRYGFCGNGTHIFSLVNAAPEGLVFLHTVLLPTQYKDVEAWSGLCVDGINGYVLRQPNLALYKEGNYYRITSRIMFEPRIPTIADFVQIENCNVTFVNISRSELQTIVPEYIDVNKTLQELSYKLPNYTVPDLVVEQYNQTILNLTSEISTLENKSAELNYTVQKLQTLIDNINSTLVDL KWLNRVET

A C-terminal trimerization domain can be added to the protomers of the229E-CoV S ectodomains trimer to promote trimerization of theectodomain.

An exemplary sequence of 229E-CoV S ectodomain including a doubleproline substitution for stabilization in the prefusion conformation,and a T4 fibritin trimerization domain is provided as SEQ ID NO: 37:

MFVLLVAYALLHIAGCQTTNGTNTSHSVCNGCVGHSENVFAVESGGYIPSNFAFNNWFLLTNTSSVVDGVVRSFQPLLLNCLWSVSGSQFTTGFVYFNGTGRGACKGFYSNASSDVIRYNINFEENLRRGTILFKTSYGAVVFYCTNNTLVSGDAHIPSGTVLGNFYCFVNTTIGNETTSAFVGALPKTVREFVISRTGHFYINGYRYFSLGDVEAVNFNVTNAATTVCTVALASYADVLVNVSQTAIANIIYCNSVINRLRCDQLSFDVPDGFYSTSPIQPVELPVSIVSLPVYHKHTFIVLYVNFEHRRGPGKCYNCRPAVINITLANFNETKGPLCVDTSHFTTQFVDNVKLARWSASINTGNCPFSFGKVNNFVKFGSVCFSLKDIPGGCAMPIMANLVNSKSHNIGSLYVSWSDGDVITGVPKPVEGVSSFMNVTLNKCTKYNIYDVSGVGVIRISNDTFLNGITYTSTSGNLLGFKDVTNGTIYSITPCNPPDQLVVYQQAVVGAMLSENFTSYGFSNVVEMPKFFYASNGTYNCTDAVLTYSSFGVCADGSIIAVQPRNVSYDSVSAIVTANLSIPFNWTTSVQVEYLQITSTPIVVDCSTYVCNGNVRCVELLKQYTSACKTIEDALRNSAMLESADVSEMLTFDKKAFTLANVSSFGDYNLSSVIPSLPRSGSRVAGRSAIEDILFSKLVTSGLGTVDADYKKCTKGLSIADLACAQYYNGIMVLPGVADAERMAMYTGSLIGGIALGGLTSAASIPFSLAIQSRLNYVALQTDVLQENQRILAASFNKAMTNIVDAFTGVNDAITQTSQALQTVATALNKIQDVVNQQGNSLNHLTSQLRQNFQAISSSIQAIYDRLDPPQADQQVDRLITGRLAALNVFVSHTLTKYTEVRASRQLAQQKVNECVKSQSKRYGFCGNGTHIFSLVNAAPEGLVFLHTVLLPTQYKDVEAWSGLCVDGINGYVLRQPNLALYKEGNYYRITSRIMFEPRIPTIADFVQIENCNVTFVNISRSELQTIVPEYIDVNKTLQELSYKLPNYTVPDLVVEQYNQTILNLTSEISTLENKSAELNYTVQKLQTLIDNINSTLVDLKWLNRVETGGYIPEAPRDGQAYVRKDGEWVLLSTF

In some embodiments, the recombinant 229E-CoV S ectodomain trimercomprises protomers comprising the ectodomain sequence of SEQ ID NO: 21.In some embodiments, the recombinant 229E-CoV S ectodomain trimercomprises protomers comprising residues 17-1108 of SEQ ID NO: 21 orresidues 17-1135 of SEQ ID NO: 37. In some embodiments, the recombinant229E-CoV S ectodomain trimer comprises protomers comprising anectodomain sequence at least 90% identical to the ectodomain sequence ofSEQ ID NO: 21, wherein the 229E-CoV S ectodomain trimer is stabilized inthe prefusion conformation and comprises the “2P” substitution and/ormodifications to remove the S1/S2 cleavage site and the S2′ cleavagesite of the protomers. In some embodiments, the recombinant 229E-CoV Sectodomain trimer comprises protomers comprising an amino acid sequenceat least 90% identical to residues 17-1108 of SEQ ID NO: 21 or residues17-1135 of SEQ ID NO: 37, wherein the 229E-CoV S ectodomain trimer isstabilized in the prefusion conformation and comprises the “2P”substitution and/or modifications to remove the S1/S2 cleavage site andthe S2′ cleavage site of the protomers.

I. PEDV-CoV

In some embodiments, the immunogen comprises a recombinant PEDV-CoV Sectodomain trimer comprising protomers comprising one or more (such astwo, for example two consecutive) proline substitutions at or near theboundary between a HR1 domain and a central helix domain that stabilizethe S ectodomain trimer in the prefusion conformation. In some suchembodiments, the one or more (such as two, for example two consecutive)proline substitutions that stabilize the S ectodomain in the prefusionconformation are located between a position 15 amino acids N-terminal ofa C-terminal residue of the HR1 and a position 5 amino acids C-terminalof a N-terminal residue of the central helix.

In some embodiments, the one or more (such as two, for example twoconsecutive) proline substitutions that stabilize the PEDV-CoV Sectodomain trimer in the prefusion conformation are located betweenresidues 1059 to 1079 (such as between residues 1069 to 1079 or betweenresidues 1073 to 1079) of the S ectodomain protomers in the trimer. Insome embodiments, the PEDV-CoV S ectodomain trimer is stabilized in theprefusion conformation by I1076P and L1077P substitutions (“2P”) in theS ectodomain protomers in the trimer. The amino acid numbering forPEDV-CoV S proteins is with reference to the PEDV-CoV S sequenceprovided as SEQ ID NO: 38.

In some embodiments, the recombinant PEDV-CoV S ectodomain trimerstabilized in the prefusion conformation comprises single-chain Sectodomain protomers comprising mutations to the S1/S2 and/or S2′protease cleavage sites to prevent protease cleavage at these sites.

In some embodiments, the protomers of the recombinant PEDV-CoV Sectodomain trimer stabilized in the prefusion conformation by the one ormore proline substitutions (such as I1076P and L1077P substitutions)comprises additional modifications for stabilization in the prefusionconformation.

With reference to the PEDV-CoV S protein sequence provided as SEQ ID NO:38, the ectodomain of the PEDV-CoV S protein includes about residues21-1322. Residues 1-20 are the signal peptide, which is removed duringcellular processing. The S1/S2 cleavage site is located at aboutposition 736/737. The S2′ cleavage site is located at about position743/744. The HR1 is located at about residues 991-1073. The centralhelix is located at about residues 1078-1119. The HR2 is located atabout 1277-1308. The C-terminal end of the S2 ectodomain is located atabout residue 1322. In some embodiments, the protomers of theprefusion-stabilized PEDV-CoV S ectodomain trimer can have a C-terminalresidue (which can be linked to a trimerization domain, or atransmembrane domain, for example) of the C-terminal residue of the HR2(e.g., position 1308), or the ectodomain (e.g., position 1322), or fromone of positions 1308-1322. The position numbering of the S protein mayvary between PEDV-CoV stains, but the sequences can be aligned todetermine relevant structural domains and cleavage sites. It will beappreciated that a few residues (such as up to 10) on the N andC-terminal ends of the ectodomain can be removed or modified in thedisclosed immunogens without decreasing the utility of the S ectodomaintrimer as an immunogen.

Exemplary PEDV-CoV S protein sequences are provided below. The prefusionstabilizing substitutions disclosed herein (and other modifications,such as substitutions to generate a single chain) can be incorporatedinto PEDV-CoV S protein sequences.

An exemplary sequence of PEDV-CoV S protein (including the ectodomainand TM and CT domains) is provided as SEQ ID NO: 38 (GenBank GI:AHZ94887.1, incorporated by reference herein):

MKSLTYFWLFLPVLSTLSLPQDVTRCSANTNFRRFFSKFNVQAPAVVVLGGYLPIGENQGVNSTWYCAGQHPTASGVHGIFVSHIRGGHGFEIGISQEPFDPSGYQLYLHKATNGNTNATARLRICQFPSIKTLGPTANNDVTTGRNCLFNKAIPAHMSEHSVVGITWDNDRVTVFSDKIYYFYFKNDWSRVATKCYNSGGCAMQYVYEPTYYMLNVTSAGEDGISYQPCTANCIGYAANVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQPLLVNCLLAIPKIYGLGQFFSFNQTIDGVCNGAAVQRAPEALRFNINDISVILAEGSIVLHTALGTNFSFVCSNSSNPHLATFAIPLGATQVPYYCFFKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQGTAIQRILYCDDPVSQLKCSQVAFDLDDGFYTISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSSSTFNSTRELPGFFYHSNDGSNCTEPVLVYSNIGVCKSGSIGYVPSQSGQVKIAPTVTGNISIPTNFSMSIRTEYLQLYNTPVSVDCATYVCNGNSRCKQLLTQYTAACKTIESALQLSARLESVEVNSMLTISDEALQLATISSFNGDGYNFTNVLGVSVYDPASRRVVQKRSFIEDLLFNKVVTNGLGTVDEDYKRCSNGRSVADLVCAQYYSGVMVLPGVVDAEKLHMYSASLIGGMVLGGFTSAAALPFSYAVQARLNYLALQTDVLQRNQQLLAESFNSAIGNITSAFESVKEAISQTSKGLNTVAHALTKVQEVVNSQGAALTQLTVQLQHNFQAISSSIDDIYSRLDILSADAQVDRLITGRLSALNAFVAQTLTKYTEVQASRKLAQQKVNECVKSQSQRYGFCGGDGEHIFSLVQAAPQGLLFLHTVLVPSDFVDVIAIAGLCVNDEIALTLREPGLVLFTHELQNHTATEYFVSSRRMFEPRKPTVSDFVQIESCVVTYVNLTRDQLPDVIPDYIDVNKTLYEILASLPNRTGPSLPLDVFNATYLNLTGEIADLEQRSESLRNTTEELQSLIYNINNTLVDLEWLNRVETYIKWPWWVWLIIFIVLIFVVSLLVFCCISTGCCGCCGCCCACFSGCCRGPRLQPYEVFEKVHVQ

An exemplary sequence of PEDV-CoV S ectodomain including a doubleproline substitution for stabilization in the prefusion conformation isprovided as SEQ ID NO: 39:

MKSLTYFWLFLPVLSTLSLPQDVTRCSANTNFRRFFSKFNVQAPAVVVLGGYLPIGENQGVNSTWYCAGQHPTASGVHGIFVSHIRGGHGFEIGISQEPFDPSGYQLYLHKATNGNTNATARLRICQFPSIKTLGPTANNDVTTGRNCLFNKAIPAHMSEHSVVGITWDNDRVTVFSDKIYYFYFKNDWSRVATKCYNSGGCAMQYVYEPTYYMLNVTSAGEDGISYQPCTANCIGYAANVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQPLLVNCLLAIPKIYGLGQFFSFNQTIDGVCNGAAVQRAPEALRFNINDISVILAEGSIVLHTALGTNFSFVCSNSSNPHLATFAIPLGATQVPYYCFFKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQGTAIQRILYCDDPVSQLKCSQVAFDLDDGFYTISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSSSTFNSTRELPGFFYHSNDGSNCTEPVLVYSNIGVCKSGSIGYVPSQSGQVKIAPTVTGNISIPTNFSMSIRTEYLQLYNTPVSVDCATYVCNGNSRCKQLLTQYTAACKTIESALQLSARLESVEVNSMLTISDEALQLATISSFNGDGYNFTNVLGVSVYDPASRRVVQKRSFIEDLLFNKVVTNGLGTVDEDYKRCSNGRSVADLVCAQYYSGVMVLPGVVDAEKLHMYSASLIGGMVLGGFTSAAALPFSYAVQARLNYLALQTDVLQRNQQLLAESFNSAIGNITSAFESVKEAISQTSKGLNTVAHALTKVQEVVNSQGAALTQLTVQLQHNFQAISSSIDDIYSRLDPPSADAQVDRLITGRLSALNAFVAQTLTKYTEVQASRKLAQQKVNECVKSQSQRYGFCGGDGEHIFSLVQAAPQGLLFLHTVLVPSDFVDVIAIAGLCVNDEIALTLREPGLVLFTHELQNHTATEYFVSSRRMFEPRKPTVSDFVQIESCVVTYVNLTRDQLPDVIPDYIDVNKTLYEILASLPNRTGPSLPLDVFNATYLNLTGEIADLEQRSESLRNTTEELQSLIYNINNTLVDLEWLNRVET

A C-terminal trimerization domain can be added to the protomers of thePEDV-CoV S ectodomains trimer to promote trimerization of theectodomain.

An exemplary sequence of PEDV-CoV S ectodomain including a doubleproline substitution for stabilization in the prefusion conformation,and a T4 fibritin trimerization domain is provided as SEQ ID NO: 40:

MKSLTYFWLFLPVLSTLSLPQDVTRCSANTNFRRFFSKFNVQAPAVVVLGGYLPIGENQGVNSTWYCAGQHPTASGVHGIFVSHIRGGHGFEIGISQEPFDPSGYQLYLHKATNGNTNATARLRICQFPSIKTLGPTANNDVTTGRNCLFNKAIPAHMSEHSVVGITWDNDRVTVFSDKIYYFYFKNDWSRVATKCYNSGGCAMQYVYEPTYYMLNVTSAGEDGISYQPCTANCIGYAANVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQPLLVNCLLAIPKIYGLGQFFSFNQTIDGVCNGAAVQRAPEALRFNINDISVILAEGSIVLHTALGTNFSFVCSNSSNPHLATFAIPLGATQVPYYCFFKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQGTAIQRILYCDDPVSQLKCSQVAFDLDDGFYTISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSSSTFNSTRELPGFFYHSNDGSNCTEPVLVYSNIGVCKSGSIGYVPSQSGQVKIAPTVTGNISIPTNFSMSIRTEYLQLYNTPVSVDCATYVCNGNSRCKQLLTQYTAACKTIESALQLSARLESVEVNSMLTISDEALQLATISSFNGDGYNFTNVLGVSVYDPASRRVVQKRSFIEDLLFNKVVTNGLGTVDEDYKRCSNGRSVADLVCAQYYSGVMVLPGVVDAEKLHMYSASLIGGMVLGGFTSAAALPFSYAVQARLNYLALQTDVLQRNQQLLAESFNSAIGNITSAFESVKEAISQTSKGLNTVAHALTKVQEVVNSQGAALTQLTVQLQHNFQAISSSIDDIYSRLDPPSADAQVDRLITGRLSALNAFVAQTLTKYTEVQASRKLAQQKVNECVKSQSQRYGFCGGDGEHIFSLVQAAPQGLLFLHTVLVPSDFVDVIAIAGLCVNDEIALTLREPGLVLFTHELQNHTATEYFVSSRRMFEPRKPTVSDFVQIESCVVTYVNLTRDQLPDVIPDYIDVNKTLYEILASLPNRTGPSLPLDVFNATYLNLTGEIADLEQRSESLRNTTEELQSLIYNINNTLVDLEWLNRVETGGYIPEAPRDGQAYVRKDGEWVLLSTF

In some embodiments, the recombinant PEDV-CoV S ectodomain trimercomprises protomers comprising the ectodomain sequence of SEQ ID NO: 39.In some embodiments, the recombinant PEDV-CoV S ectodomain trimercomprises protomers comprising residues 21-1322 of SEQ ID NO: 39 orresidues 21-1349 of SEQ ID NO: 40. In some embodiments, the recombinantPEDV-CoV S ectodomain trimer comprises protomers comprising anectodomain sequence at least 90% identical to the ectodomain sequence ofSEQ ID NO: 39, wherein the PEDV-CoV S ectodomain trimer is stabilized inthe prefusion conformation and comprises the “2P” substitution and/ormodifications to remove the S1/S2 cleavage site and the S2′ cleavagesite of the protomers. In some embodiments, the recombinant PEDV-CoV Sectodomain trimer comprises protomers comprising an amino acid sequenceat least 90% identical to residues 21-1322 of SEQ ID NO: 39 or residues21-1349 of SEQ ID NO: 40, wherein the PEDV-CoV S ectodomain trimer isstabilized in the prefusion conformation and comprises the “2P”substitution and/or modifications to remove the S1/S2 cleavage site andthe S2′ cleavage site of the protomers.

I. SDCV

In some embodiments, the immunogen comprises a recombinant swine deltacoronavirus (SDCV) S ectodomain trimer comprising protomers comprisingone or more (such as two, for example two consecutive) prolinesubstitutions at or near the boundary between a HR1 domain and a centralhelix domain that stabilize the S ectodomain trimer in the prefusionconformation. In some such embodiments, the one or more (such as two,for example two consecutive) proline substitutions that stabilize the Sectodomain in the prefusion conformation are located between a position15 amino acids N-terminal of a C-terminal residue of the HR1 and aposition 5 amino acids C-terminal of a N-terminal residue of the centralhelix.

In some embodiments, the one or more (such as two, for example twoconsecutive) proline substitutions that stabilize the SDCV S ectodomaintrimer in the prefusion conformation are located between residues 838 to858 (such as between residues 848 to 858 or between residues 854 to 858)of the S ectodomain protomers in the trimer. In some embodiments, theSDCV S ectodomain trimer is stabilized in the prefusion conformation byE855P and V856P substitutions (“2P”) in the S ectodomain protomers inthe trimer. The amino acid numbering for SDCV S proteins is withreference to the SDCV S sequence provided as SEQ ID NO: 41.

In some embodiments, the recombinant SDCV S ectodomain trimer stabilizedin the prefusion conformation comprises single-chain S ectodomainprotomers comprising mutations to the S1/S2 and/or S2′ protease cleavagesites to prevent protease cleavage at these sites.

In some embodiments, the protomers of the recombinant SDCV S ectodomaintrimer stabilized in the prefusion conformation by the one or moreproline substitutions (such as E855P and V856P substitutions) comprisesadditional modifications for stabilization in the prefusionconformation.

With reference to the SDCV S protein sequence provided as SEQ ID NO: 41,the ectodomain of the SDCV S protein includes about residues 20-1093.Residues 1-19 are the signal peptide, which is removed during cellularprocessing. The HR1 is located at about residues 770-854. The centralhelix is located at about residues 857-898. The HR2 is located at about1034-1079. The C-terminal end of the S2 ectodomain is located at aboutresidue 1093. In some embodiments, the protomers of theprefusion-stabilized SDCV S ectodomain trimer can have a C-terminalresidue (which can be linked to a trimerization domain, or atransmembrane domain, for example) of the C-terminal residue of the HR2(e.g., position 1079), or the ectodomain (e.g., position 1093), or fromone of positions 1079-1093. The position numbering of the S protein mayvary between SDCV stains, but the sequences can be aligned to determinerelevant structural domains and cleavage sites. It will be appreciatedthat a few residues (such as up to 10) on the N and C-terminal ends ofthe ectodomain can be removed or modified in the disclosed immunogenswithout decreasing the utility of the S ectodomain trimer as animmunogen.

Exemplary SDCV S protein sequences are provided below. The prefusionstabilizing substitutions disclosed herein (and other modifications,such as substitutions to generate a single chain) can be incorporatedinto SDCV S protein sequences.

An exemplary sequence of SDCV S protein (including the ectodomain and TMand CT domains) is provided as SEQ ID NO: 41 (GenBank GI: AMN91621.1,incorporated by reference herein):

MQRALLIMTLLCLARAKFADDLLDLLTFPGAHRFLHKPTRNDSILYSRANNNFDVGVLPGYPTKNVNLFSPLTNSTLPINGLHRSYQPLMLNCLTKITNQTLSMYLQPSEIQTYSCGGAMVKYQTHDAVRIILDLIATDRISVEVVGQAGENYVFVCSDQFNYTTALHNSTFFSLNSQLYCFTNNTYLGILPPDLTDFTVYRTGQFYANGYLLGTLPITVNYVRLYRGQLSANSAHFALANLTDTLITLTNTTISQITYCDKSVVDSIACQRSSHQVEDGFYSDPKSAVRARQRTIVTLPKLPELEVVQLNISAHMDFGEARLDSVTINGNTSYCVTKPYFRLETNFLCRGCTMNLRTDTCSFDLSAVNNGMSFSQFCLSTESGACEMKIIVTYVWNYLLRQRLYVTAVEGQTHTGTTSVHATDTSSVITDVCTDYTIYGVSGTGIIKPSDLLLHNGIAFTSPTGELYAFKNITTGKTLQVLPCETPSQLIVINNTVVGAITSSNSTENNRFTTTIVTPTFFYSTNATTLNCTKPVLSYGPISVCSDGAIAGTSTLQNTRPSIVSLYDGEIEIPSAFSLSVQTEYLQVQAEQVIVDCPQYVCNGNSRCLQLLAQYTSACSNIEVALHSSAQLDSREIISMFKTSTQSLQLANITNFKGDYNFSSILTSRVGGRSAIEDLLFNKVVTSGLGTVDQDYKSCSRNMAIADLVCSQYYNGIMVLPGVVDAEKMAMYTGSLTGAMVFGGLTAAAAIPFATAVQARLNYVALQTNVLQENQKILAESFNQAVGNISLALSSVNDAIQQTSEALNTVAIAIKKIQTVVNQQGEALSHLTAQLSNNFQAISTSIQDIYNRLEEVEANQQVDRLINGRLAALNAYVTQLLNQMSQIRQSRLLAQQKINECVKSQSPRYGFCGNGTHIFSLTQTAPNGIFFMHAVLVPNKFTRVNASAGICVDNTRGYSLQPQLILYQFNNSWRVTPRNMYEPRLPRQADFIQLTDCSVTFYNTTAANLPNIIPDVIDVNQTVSDIIDNLPTATPPQWDVGIYNNTILNLTVEINDLQERSKNLSQIADRLQNYIDNLNNTLVDLEWLNRVETYLKWPWYIWLAIALALIAFVTILITIFLCTGCCGGCFGCCGGCFGLFSKKKRYTDDQ PTPSFKFKEW

An exemplary sequence of SDCV S ectodomain including a double prolinesubstitution for stabilization in the prefusion conformation is providedas SEQ ID NO: 42:

MQRALLIMTLLCLARAKFADDLLDLLTFPGAHRFLHKPTRNDSILYSRANNNFDVGVLPGYPTKNVNLFSPLTNSTLPINGLHRSYQPLMLNCLTKITNQTLSMYLQPSEIQTYSCGGAMVKYQTHDAVRIILDLIATDRISVEVVGQAGENYVFVCSDQFNYTTALHNSTFFSLNSQLYCFTNNTYLGILPPDLTDFTVYRTGQFYANGYLLGTLPITVNYVRLYRGQLSANSAHFALANLTDTLITLTNTTISQITYCDKSVVDSIACQRSSHQVEDGFYSDPKSAVRARQRTIVTLPKLPELEVVQLNISAHMDFGEARLDSVTINGNTSYCVTKPYFRLETNFLCRGCTMNLRTDTCSFDLSAVNNGMSFSQFCLSTESGACEMKIIVTYVWNYLLRQRLYVTAVEGQTHTGTTSVHATDTSSVITDVCTDYTIYGVSGTGIIKPSDLLLHNGIAFTSPTGELYAFKNITTGKTLQVLPCETPSQLIVINNTVVGAITSSNSTENNRFTTTIVTPTFFYSTNATTLNCTKPVLSYGPISVCSDGAIAGTSTLQNTRPSIVSLYDGEIEIPSAFSLSVQTEYLQVQAEQVIVDCPQYVCNGNSRCLQLLAQYTSACSNIEVALHSSAQLDSREIISMFKTSTQSLQLANITNFKGDYNFSSILTSRVGGRSAIEDLLFNKVVTSGLGTVDQDYKSCSRNMAIADLVCSQYYNGIMVLPGVVDAEKMAMYTGSLTGAMVFGGLTAAAAIPFATAVQARLNYVALQTNVLQENQKILAESFNQAVGNISLALSSVNDAIQQTSEALNTVAIAIKKIQTVVNQQGEALSHLTAQLSNNFQAISTSIQDIYNRLEPPEANQQVDRLINGRLAALNAYVTQLLNQMSQIRQSRLLAQQKINECVKSQSPRYGFCGNGTHIFSLTQTAPNGIFFMHAVLVPNKFTRVNASAGICVDNTRGYSLQPQLILYQFNNSWRVTPRNMYEPRLPRQADFIQLTDCSVTFYNTTAANLPNIIPDVIDVNQTVSDIIDNLPTATPPQWDVGIYNNTILNLTVEINDLQERSKNLSQIADRLQNYIDNLNNTLVDLEWLNRVET

A C-terminal trimerization domain can be added to the protomers of theSDCV S ectodomains trimer to promote trimerization of the ectodomain.

An exemplary sequence of SDCV S ectodomain including a double prolinesubstitution for stabilization in the prefusion conformation, and a T4fibritin trimerization domain is provided as SEQ ID NO: 43:

MQRALLIMTLLCLARAKFADDLLDLLTFPGAHRFLHKPTRNDSILYSRANNNFDVGVLPGYPTKNVNLFSPLTNSTLPINGLHRSYQPLMLNCLTKITNQTLSMYLQPSEIQTYSCGGAMVKYQTHDAVRIILDLIATDRISVEVVGQAGENYVFVCSDQFNYTTALHNSTFFSLNSQLYCFTNNTYLGILPPDLTDFTVYRTGQFYANGYLLGTLPITVNYVRLYRGQLSANSAHFALANLTDTLITLTNTTISQITYCDKSVVDSIACQRSSHQVEDGFYSDPKSAVRARQRTIVTLPKLPELEVVQLNISAHMDFGEARLDSVTINGNTSYCVTKPYFRLETNFLCRGCTMNLRTDTCSFDLSAVNNGMSFSQFCLSTESGACEMKIIVTYVWNYLLRQRLYVTAVEGQTHTGTTSVHATDTSSVITDVCTDYTIYGVSGTGIIKPSDLLLHNGIAFTSPTGELYAFKNITTGKTLQVLPCETPSQLIVINNTVVGAITSSNSTENNRFTTTIVTPTFFYSTNATTLNCTKPVLSYGPISVCSDGAIAGTSTLQNTRPSIVSLYDGEIEIPSAFSLSVQTEYLQVQAEQVIVDCPQYVCNGNSRCLQLLAQYTSACSNIEVALHSSAQLDSREIISMFKTSTQSLQLANITNFKGDYNFSSILTSRVGGRSAIEDLLFNKVVTSGLGTVDQDYKSCSRNMAIADLVCSQYYNGIMVLPGVVDAEKMAMYTGSLTGAMVFGGLTAAAAIPFATAVQARLNYVALQTNVLQENQKILAESFNQAVGNISLALSSVNDAIQQTSEALNTVAIAIKKIQTVVNQQGEALSHLTAQLSNNFQAISTSIQDIYNRLEPPEANQQVDRLINGRLAALNAYVTQLLNQMSQIRQSRLLAQQKINECVKSQSPRYGFCGNGTHIFSLTQTAPNGIFFMHAVLVPNKFTRVNASAGICVDNTRGYSLQPQLILYQFNNSWRVTPRNMYEPRLPRQADFIQLTDCSVTFYNTTAANLPNIIPDVIDVNQTVSDIIDNLPTATPPQWDVGIYNNTILNLTVEINDLQERSKNLSQIADRLQNYIDNLNNTLVDLEWLNRVETGGYIPEA PRDGQAYVRKDGEWVLLSTF

In some embodiments, the recombinant SDCV S ectodomain trimer comprisesprotomers comprising the ectodomain sequence of SEQ ID NO: 42. In someembodiments, the recombinant SDCV S ectodomain trimer comprisesprotomers comprising residues 20-1093 of SEQ ID NO: 42 or residues20-1120 of SEQ ID NO: 43. In some embodiments, the recombinant SDCV Sectodomain trimer comprises protomers comprising an ectodomain sequenceat least 90% identical to the ectodomain sequence of SEQ ID NO: 39,wherein the SDCV S ectodomain trimer is stabilized in the prefusionconformation and comprises the “2P” substitution and/or modifications toremove the S1/S2 cleavage site and the S2′ cleavage site of theprotomers. In some embodiments, the recombinant SDCV S ectodomain trimercomprises protomers comprising an amino acid sequence at least 90%identical to residues 20-1093 of SEQ ID NO: 39 or residues 20-1120 ofSEQ ID NO: 43, wherein the SDCV S ectodomain trimer is stabilized in theprefusion conformation and comprises the “2P” substitution and/ormodifications to remove the S1/S2 cleavage site and the S2′ cleavagesite of the protomers.

J. Single Chain S Proteins

In some embodiments, the CoV S ectodomain trimer can be composed ofthree single-chain CoV S ectodomain protomers, each including a singlepolypeptide chain including the 51 protein and S2 ectodomain. Singlechain CoV S ectodomain protomers can be generated by mutating the S1/S2and S2′ protease cleavage sites to prevent cleavage and formation ofdistinct 51 and S2 polypeptide chains. In some embodiments, the 51 andS2 polypeptides in the single chain CoV S ectodomain protomers arejoined by a linker, such as a peptide linker. Examples of peptidelinkers that can be used include glycine, serine, and glycine-serinelinkers. Any of the stabilizing mutations (or combinations thereof)disclosed herein can be included in the single chain coronavirus Sectodomain protomers as long as the coronavirus S ectodomain trimercomposed of such protomers retains the desired properties (e.g., theprefusion conformation).

K. Linkage to a Trimerization Domain

In several embodiments, the S ectodomain protomers in the disclosedcoronavirus S ectodomain trimers can be linked at their C-terminus(C-terminal linkage) to a trimerization domain to promote trimerizationof the S ectodomain protomers, and to stabilize the membrane proximalaspect of the recombinant S ectodomains in a trimeric configuration.

Non-limiting examples of exogenous multimerization domains that promotestable trimers of soluble recombinant proteins include: the GCN4 leucinezipper (Harbury et al. 1993 Science 262:1401-1407), the trimerizationmotif from the lung surfactant protein (Hoppe et al. 1994 FEBS Lett344:191-195), collagen (McAlinden et al. 2003 J Biol Chem278:42200-42207), and the phage T4 fibritin Foldon (Miroshnikov et al.1998 Protein Eng 11:329-414), any of which can be linked to arecombinant coronavirus S ectodomain described herein (e.g., by linkageto the C-terminus of S2) to promote trimerization of the recombinantcoronavirus S ectodomain.

In some examples, the C-terminus of the S2 subunit of the S ectodomaincan be linked to a T4 fibritin Foldon domain. In specific examples, theT4 fibritin Foldon domain can include the amino acid sequenceGYIPEAPRDGQAYVRKDGEWVLLSTF (SEQ ID NO: 27), which adopts a β-propellerconformation, and can fold and trimerize in an autonomous way (Tao etal. 1997 Structure 5:789-798). Optionally, the heterologoustrimerization is connected to the recombinant coronavirus S ectodomainvia a peptide linker, such as an amino acid linker. Non-limitingexamples of peptide linkers that can be used include glycine, serine,and glycine-serine linkers.

L. Membrane Anchored Embodiments

In some embodiments, the coronavirus S ectodomain trimer can be membraneanchored, for example, for embodiments where the coronavirus Sectodomain trimer is expressed on an attenuated viral vaccine, or avirus like particle. In such embodiments, the protomers in the trimertypically each comprise a C-terminal linkage to a transmembrane domain,such as the transmembrane domain (and optionally the cytosolic tail) ofcorresponding coronavirus. For example, the protomers of a disclosedSARS-CoV S ectodomain trimer can be linked to a SARS-CoV S transmembraneand cytosolic tail. In some embodiments, one or more peptide linkers(such as a gly-ser linker, for example, a 10 amino acid glycine-serinepeptide linker can be used to link the recombinant S ectodomain protomerto the transmembrane domain. The protomers linked to the transmembranedomain can include any of the stabilizing mutations provided herein (orcombinations thereof) as long as the recombinant coronavirus Sectodomain trimer formed from the protomers linked to the transmembranedomain retains the desired properties (e.g., the coronavirus S prefusionconformation).

M. Additional Description

The coronavirus S protein or fragments thereof can be produced usingrecombinant techniques, or chemically or enzymatically synthesized.

Analogs and variants of the coronavirus S protein or fragments thereofmay be used in the methods and systems of the present invention. Throughthe use of recombinant DNA technology, variants of the coronavirus Sprotein or fragments thereof may be prepared by altering the underlyingDNA. All such variations or alterations in the structure of thecoronavirus S ectodomain or fragments thereof resulting in variants areincluded within the scope of this invention. Such variants includeinsertions, substitutions, or deletions of one or more amino acidresidues, glycosylation variants, unglycosylated coronavirus Sectodomain or fragments thereof, organic and inorganic salts, covalentlymodified derivatives of the coronavirus S protein or fragments thereof,or a precursor thereof. Such variants may maintain one or more of thefunctional, biological activities of the coronavirus S protein orfragment thereof, such as binding to cell surface receptor. Thecoronavirus S protein or a fragment thereof can be modified, forexample, by PEGylation, to increase the half-life of the protein in therecipient, to retard clearance from the pericardial space, and/or tomake the protein more stable for delivery to a subject.

In some embodiments, a coronavirus S protein or fragment thereof usefulwithin the disclosure is modified to produce peptide mimetics byreplacement of one or more naturally occurring side chains of the 20genetically encoded amino acids (or D-amino acids) with other sidechains, for example with groups such as alkyl, lower alkyl, cyclic 4-,5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(loweralkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivativesthereof, and with 4-, 5-, 6-, to 7-membered heterocyclics. For example,proline analogs can be made in which the ring size of the prolineresidue is changed from a 5-membered ring to a 4-, 6-, or 7-memberedring. Cyclic groups can be saturated or unsaturated, and if unsaturated,can be aromatic or non-aromatic. Heterocyclic groups can contain one ormore nitrogen, oxygen, and/or sulphur heteroatoms. Examples of suchgroups include furazanyl, furyl, imidazolidinyl, imidazolyl,imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino),oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g.,1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl(e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl,thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl groups.These heterocyclic groups can be substituted or unsubstituted. Where agroup is substituted, the substituent can be alkyl, alkoxy, halogen,oxygen, or substituted or unsubstituted phenyl. Peptides, as well aspeptide analogs and mimetics, can also be covalently bound to one ormore of a variety of nonproteinaceous polymers, for example,polyethylene glycol, polypropylene glycol, or polyoxyalkenes, asdescribed in U.S. Pat. Nos. 4,640,835; 4,496,668; 4,301,144; 4,668,417;4,791,192; and 4,179,337.

N. Protein Nanoparticles

In some embodiments a protein nanoparticle is provided that includes oneor more of the disclosed recombinant coronavirus S ectodomain trimers(e.g., a MERS-CoV S ectodomain trimer or a SARS-CoV S ectodomaintrimer). Non-limiting example of nanoparticles include ferritinnanoparticles, encapsulin nanoparticles, Sulfur Oxygenase Reductase(SOR) nanoparticles, and lumazine synthase nanoparticles, which arecomprised of an assembly of monomeric subunits including ferritinproteins, encapsulin proteins, SOR proteins, and lumazine synthase,respectively. Additional protein nanoparticle structures are describedby Heinze et al., J Phys Chem B., 120(26):5945-52, 2016; Hsia et al.,Nature, 535(7610):136-9, 2016; and King et al., Nature, 510(7503):103-8,2014; each of which is incorporated by reference herein. To constructsuch protein nanoparticles a protomer of the coronavirus S ectodomaintrimer can be linked to a subunit of the protein nanoparticle (such as aferritin protein, an encapsulin protein, a SOR protein, or a lumazinesynthase protein) and expressed in cells under appropriate conditions.The fusion protein self-assembles into a nanoparticle any can bepurified.

In some embodiments, a protomer of a disclosed recombinant coronavirus Sectodomain trimer (e.g., a MERS-CoV S ectodomain trimer or a SARS-CoV Sectodomain trimer) can be linked to a ferritin subunit to construct aferritin nanoparticle. Ferritin nanoparticles and their use forimmunization purposes (e.g., for immunization against influenzaantigens) have been disclosed in the art (see, e.g., Kanekiyo et al.,Nature, 499:102-106, 2013, incorporated by reference herein in itsentirety). Ferritin is a globular protein that is found in all animals,bacteria, and plants, and which acts primarily to control the rate andlocation of polynuclear Fe(III)₂O₃ formation through the transportationof hydrated iron ions and protons to and from a mineralized core. Theglobular form of the ferritin nanoparticle is made up of monomericsubunits, which are polypeptides having a molecule weight ofapproximately 17-20 kDa. An example of the amino acid sequence of onesuch monomeric ferritin subunit is represented by:

(SEQ ID NO: 23) ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

Each monomeric subunit has the topology of a helix bundle which includesa four antiparallel helix motif, with a fifth shorter helix (thec-terminal helix) lying roughly perpendicular to the long axis of the 4helix bundle. According to convention, the helices are labeled ‘A, B, C,D & E’ from the N-terminus respectively. The N-terminal sequence liesadjacent to the capsid three-fold axis and extends to the surface, whilethe E helices pack together at the four-fold axis with the C-terminusextending into the capsid core. The consequence of this packing createstwo pores on the capsid surface. It is expected that one or both ofthese pores represent the point by which the hydrated iron diffuses intoand out of the capsid. Following production, these monomeric subunitproteins self-assemble into the globular ferritin protein. Thus, theglobular form of ferritin comprises 24 monomeric, subunit proteins, andhas a capsid-like structure having 432 symmetry. Methods of constructingferritin nanoparticles are known to the person of ordinary skill in theart and are further described herein (see, e.g., Zhang, Int. J. Mol.Sci., 12:5406-5421, 2011, which is incorporated herein by reference inits entirety).

In specific examples, the ferritin polypeptide is E. coli ferritin,Helicobacter pylori ferritin, human light chain ferritin, bullfrogferritin or a hybrid thereof, such as E. coli-human hybrid ferritin, E.coli-bullfrog hybrid ferritin, or human-bullfrog hybrid ferritin.Exemplary amino acid sequences of ferritin polypeptides and nucleic acidsequences encoding ferritin polypeptides for use to make a ferritinnanoparticle including a recombinant coronavirus S ectodomain can befound in GENBANK®, for example at accession numbers ZP_03085328,ZP_06990637, EJB64322.1, AAA35832, NP_000137 AAA49532, AAA49525,AAA49524 and AAA49523, which are specifically incorporated by referenceherein in their entirety as available Apr. 10, 2015. In someembodiments, a recombinant coronavirus S ectodomain can be linked to aferritin subunit including an amino acid sequence at least 80% (such asat least 85%, at least 90%, at least 95%, or at least 97%) identical toamino acid sequence set forth as SEQ ID NO: 122.

In some embodiments, a protomer of a disclosed recombinant coronavirus Sectodomain trimer (e.g., a MERS-CoV S ectodomain trimer or a SARS-CoV Sectodomain trimer) can be linked to a lumazine synthase subunit toconstruct a lumazine synthase nanoparticle. The globular form oflumazine synthase nanoparticle is made up of monomeric subunits; anexample of the sequence of one such lumazine synthase subunit isprovides as the amino acid sequence set forth as:

(SEQ ID NO: 24) MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLF KSLR.

In some embodiments, a protomer of a disclosed recombinant coronavirus Sectodomain trimer can be linked to a lumazine synthase subunit includingan amino acid sequence at least 80% (such as at least 85%, at least 90%,at least 95%, or at least 97%) identical to amino acid sequence setforth as SEQ ID NO: 24.

In some embodiments, a protomer of a disclosed recombinant coronavirus Sectodomain trimer (e.g., a MERS-CoV S ectodomain trimer or a SARS-CoV Sectodomain trimer) can be linked to an encapsulin nanoparticle subunitto construct an encapsulin nanoparticle. The globular form of theencapsulin nanoparticle is made up of monomeric subunits; an example ofthe sequence of one such encapsulin subunit is provides as the aminoacid sequence set forth as

(SEQ ID NO: 25) MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETF TFQVVNPEALILLKF.

In some embodiments, a protomer of a disclosed recombinant coronavirus Sectodomain trimer can be linked to an encapsulin subunit including anamino acid sequence at least 80% (such as at least 85%, at least 90%, atleast 95%, or at least 97%) identical to amino acid sequence set forthas SEQ ID NO: 25.

Encapsulin proteins are a conserved family of bacterial proteins alsoknown as linocin-like proteins that form large protein assemblies thatfunction as a minimal compartment to package enzymes. The encapsulinassembly is made up of monomeric subunits, which are polypeptides havinga molecule weight of approximately 30 kDa. Following production, themonomeric subunits self-assemble into the globular encapsulin assemblyincluding 60, or in some cases, 180 monomeric subunits. Methods ofconstructing encapsulin nanoparticles are known to the person ofordinary skill in the art, and further described herein (see, forexample, Sutter et al., Nature Struct. and Mol. Biol., 15:939-947, 2008,which is incorporated by reference herein in its entirety). In specificexamples, the encapsulin polypeptide is bacterial encapsulin, such asThermotoga maritime or Pyrococcus furiosus or Rhodococcus erythropolisor Myxococcus xanthus encapsulin.

In some embodiments, a protomer of a disclosed recombinant coronavirus Sectodomain trimer (e.g., a MERS-CoV S ectodomain trimer or a SARS-CoV Sectodomain trimer) can be linked to a Sulfur Oxygenase Reductase (SOR)subunit to construct a recombinant SOR nanoparticle. In someembodiments, the SOR subunit can include the amino acid sequence setforth as

(SEQ ID NO: 26) MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETF TFQVVNPEALILLKF.

In some embodiments, a protomer of a disclosed recombinant coronavirus Sectodomain trimer can be linked to a SOR subunit including an amino acidsequence at least 80% (such as at least 85%, at least 90%, at least 95%,or at least 97%) identical to amino acid sequence set forth as SEQ IDNO: 26.

SOR proteins are microbial proteins (for example from thethermoacidophilic archaeon Acidianus ambivalens that form 24 subunitprotein assemblies. Methods of constructing SOR nanoparticles are knownto the person of ordinary skill in the art (see, e.g., Urich et al.,Science, 311:996-1000, 2006, which is incorporated by reference hereinin its entirety). An example of an amino acid sequence of a SOR proteinfor use to make SOR nanoparticles is set forth in Urich et al., Science,311:996-1000, 2006, which is incorporated by reference herein in itsentirety.

For production purposes, the recombinant coronavirus S ectodomain linkedto the nanoparticle subunit can include an N-terminal signal peptidethat is cleaved during cellular processing. For example, the recombinantcoronavirus S ectodomain protomer linked to the protein nanoparticlesubunit can include a signal peptide at its N-terminus including, forexample, a native coronavirus S signal peptide.

The protein nanoparticles can be expressed in appropriate cells (e.g.,HEK 293 Freestyle cells) and fusion proteins are secreted from the cellsself-assembled into nanoparticles. The nanoparticles can be purifiedusing known techniques, for example by a few different chromatographyprocedures, e.g. Mono Q (anion exchange) followed by size exclusion(SUPEROSE® 6) chromatography.

Several embodiments include a monomeric subunit of a ferritin,encapsulin, SOR, or lumazine synthase protein, or any portion thereofwhich is capable of directing self-assembly of monomeric subunits intothe globular form of the protein Amino acid sequences from monomericsubunits of any known ferritin, encapsulin, SOR, or lumazine synthaseprotein can be used to produce fusion proteins with the recombinantcoronavirus S ectodomain or immunogenic fragment thereof, so long as themonomeric subunit is capable of self-assembling into a nanoparticledisplaying the recombinant coronavirus S ectodomain or immunogenicfragment thereof on its surface.

The fusion proteins need not comprise the full-length sequence of amonomeric subunit polypeptide of a ferritin, encapsulin, SOR, orlumazine synthase protein. Portions, or regions, of the monomericsubunit polypeptide can be utilized so long as the portion comprisesamino acid sequences that direct self-assembly of monomeric subunitsinto the globular form of the protein.

III. Polynucleotides and Expression

Polynucleotides encoding a protomer of any of the disclosed recombinantS ectodomain trimers are also provided. These polynucleotides includeDNA, cDNA and RNA sequences which encode the protomer, as well asvectors including the DNA, cDNA and RNA sequences, such as a DNA or RNAvector used for immunization. The genetic code to construct a variety offunctionally equivalent nucleic acids, such as nucleic acids whichdiffer in sequence but which encode the same protein sequence, or encodea conjugate or fusion protein including the nucleic acid sequence.

An exemplary nucleic acid sequence encoding MERS-CoV S protein isprovided as SEQ ID NO: 5:

atgattcactccgtgttcctgctgatgttcctgctgactcctacagagagctatgtggatgtgggacctgattccgtcaagagcgcctgcatcgaagtggacattcagcagaccttctttgataagacatggccaagacccatcgacgtgagcaaagccgatggcatcatctaccctcaggggaggacctattccaatatcacaattacttaccagggcctgttcccatatcagggagaccacggcgatatgtacgtgtattctgctggccatgcaacagggaccacacctcagaagctgtttgtggctaactacagccaggacgtcaaacagttcgcaaatggatttgtggtccgcatcggcgccgctgcaaactctaccggcacagtgatcatttcacctagcacttccgcaaccatccgaaaaatctacccagccttcatgctgggaagctccgtgggcaattttagcgacgggaaaatgggacggttctttaaccacaccctggtgctgctgcctgatggatgcggcacactgctgagggctttctactgtatcctggagccacgcagcggaaaccactgccccgcaggaaatagctacacctcctttgccacatatcatactccagctaccgactgttccgatggcaactacaatcgaaacgcctctctgaatagtttcaaggaatacttcaacctgcggaattgcacattcatgtacacttataacatcaccgaggacgaaattctggagtggttcggaatcactcagaccgcacagggcgtgcacctgttttctagtcgctacgtcgacctgtatggcgggaacatgttccagtttgccactctgcccgtgtacgataccatcaagtactattccatcattcctcattcaatccgcagcattcagtccgatcgaaaggcttgggccgctttctacgtgtataaactgcagccactgaccttcctgctggactttagcgtcgatggctacatccggagagccattgactgcgggtttaatgatctgtcccagctgcactgttcttacgaaagtttcgacgtggagtccggcgtgtattctgtctcaagctttgaggccaagccctctgggagtgtggtcgagcaggctgaaggagtggagtgcgatttcagtcctctgctgtcagggaccccccctcaggtgtacaacttcaagcggctggtctttactaactgtaactacaatctgaccaagctgctgtcactgttcagcgtgaatgactttacatgctcccagatcagccccgcagccattgctagtaactgttactcctctctgatcctggactacttctcatatccactgagtatgaagagcgacctgagcgtgagttcagccggccccatcagccagttcaactataaacagagcttcagcaatcctacatgcctgattctggctactgtgccacataatctgactaccatcactaagcccctgaaatactcctatattaacaagtgcagccggttcctgtccgacgatagaaccgaagtgccacagctggtcaacgccaatcagtactctccctgtgtgagtatcgtcccttcaaccgtgtgggaagacggggattactatagaaaacagctgagccccctggagggaggaggatggctggtggcatccggatctacagtcgccatgactgagcagctgcagatggggttcggaatcacagtgcagtacggcacagacactaactctgtctgtcccaagctggaattcgctaacgatactaagatcgcaagtcagctgggaaactgcgtggagtactctctgtatggcgtgagtggcagaggggtcttccagaattgtaccgcagtgggcgtccgacagcagcggtttgtgtacgacgcctatcagaatctggtcggctactatagcgacgatgggaactactattgcctgagggcctgtgtgagcgtccctgtgtccgtcatctacgataaggaaaccaaaacacacgccacactgttcgggtccgtggcttgcgagcatattagctccacaatgtctcagtacagtagatcaactaggtcaatgctgaagaggcgcgatagcacctatggacctctgcagacaccagtggggtgtgtcctgggactggtgaactctagtctgtttgtcgaggactgcaagctgcccctgggccagagcctgtgcgccctgcccgacacccccagcaccctgaccccccggagcgtgcggagcgtgcccggcgagatgcggctggccagcatcgccttcaaccaccccatccaggtggaccagctgaacagcagctacttcaagctgagcatccccaccaacttcagcttcggcgtgacccaggagtacatccagaccaccatccagaaggtgaccgtggactgcaagcagtacgtgtgcaacggcttccagaagtgcgagcagctgctgcgggagtacggccagttctgcagcaagatcaaccaggccctgcacggcgccaacctgcggcaggacgacagcgtgcggaacctgttcgccagcgtgaagagcagccagagcagccccatcatccccggcttcggcggcgacttcaacctgaccctgctggagcccgtgagcatcagcaccggcagccggagcgcccggagcgccatcgaggacctgctgttcgacaaggtgaccatcgccgaccccggctacatgcagggctacgacgactgcatgcagcagggccccgccagcgcccgggacctgatctgcgcccagtacgtggccggctacaaggtgctgccccccctgatggacgtgaacatggaggccgcctacaccagcagcctgctgggcagcatcgccggcgtgggctggaccgccggcctgagcagcttcgccgccatccccttcgcccagagcatcttctaccggctgaacggcgtgggcatcacccagcaggtgctgagcgagaaccagaagctgatcgccaacaagttcaaccaggccctgggcgccatgcagaccggcttcaccaccaccaacgaggccttccacaaggtgcaggacgccgtgaacaacaacgcccaggccctgagcaagctggccagcgagctgagcaacaccttcggcgccatcagcgccagcatcggcgacatcatccagcggctggacgtgctggagcaggacgcccagatcgaccggctgatcaacggccggctgaccaccctgaacgccttcgtggcccagcagctggtgcggagcgagagcgccgccctgagcgcccagctggccaaggacaaggtgaacgagtgcgtgaaggcccagagcaagcggagcggcttctgcggccagggcacccacatcgtgagcttcgtggtgaacgcccccaacggcctgtacttcatgcacgtgggctactaccccagcaaccacatcgaggtggtgagcgcctacggcctgtgcgacgccgccaaccccaccaactgcatcgcccccgtgaacggctacttcatcaagaccaacaacacccggatcgtggacgagtggagctacaccggcagcagcttctacgcccccgagcccatcaccagcctgaacaccaagtacgtggccccccaggtgacctaccagaacatcagcaccaacctgcccccccccctgctgggcaacagcaccggcatcgacttccaggacgagctggacgagttcttcaagaacgtgagcaccagcatccccaacttcggcagcctgacccagatcaacaccaccctgctggacctgacctacgagatgctgagcctgcagcaggtggtgaaggccctgaacgagagctacatcgacctgaaggagctgggcaactacacctactacaacaagtggccctggtacatctggctgggcttcatcgccggcctggtggccctggccctgtgcgtgttcttcatcctgtgctgcaccggctgcggcaccaactgcatgggcaagctgaagtgcaaccggtgctgcgaccggtacgaggagtacgacctggagccccacaaggtg cacgtgcactgaThe DNA sequence of the MERS-CoV S protomer provided above can bemodified to introduce the amino acid substitutions and deletionsdisclosed herein for prefusion stabilization, such as the “2P”substitutions.

In several embodiments, the nucleic acid molecule encodes a precursor ofthe protomer, that, when expressed in an appropriate cell, is processedinto a disclosed coronavirus S ectodomain protomer that canself-assemble into the corresponding recombinant coronavirus Sectodomain trimer. For example, the nucleic acid molecule can encode arecombinant coronavirus S ectodomain including a N-terminal signalsequence for entry into the cellular secretory system that isproteolytically cleaved in the during processing of the recombinantcoronavirus S ectodomain in the cell.

In several embodiments, the nucleic acid molecule encodes a precursor Spolypeptide that, when expressed in an appropriate cell, is processedinto a disclosed recombinant coronavirus S ectodomain protomer includingS1 and S2 polypeptides, wherein the recombinant S ectodomain protomerincludes any of the appropriate stabilizing modifications describedherein, and optionally can be linked to a trimerization domain, such asa T4 Fibritin trimerization domain.

Exemplary nucleic acids can be prepared by cloning techniques. Examplesof appropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises areknown (see, e.g., Sambrook et al. (Molecular Cloning: A LaboratoryManual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,through supplement 104, 2013).

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

The polynucleotides encoding a disclosed recombinant coronavirus Sectodomain protomer can include a recombinant DNA which is incorporatedinto a vector (such as an expression vector) into an autonomouslyreplicating plasmid or virus or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (such as a cDNA)independent of other sequences. The nucleotides can be ribonucleotides,deoxyribonucleotides, or modified forms of either nucleotide. The termincludes single and double forms of DNA.

Polynucleotide sequences encoding a disclosed recombinant coronavirus Sectodomain protomer can be operatively linked to expression controlsequences. An expression control sequence operatively linked to a codingsequence is ligated such that expression of the coding sequence isachieved under conditions compatible with the expression controlsequences. The expression control sequences include, but are not limitedto, appropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons.

DNA sequences encoding the disclosed recombinant S ectodomain protomercan be expressed in vitro by DNA transfer into a suitable host cell. Thecell may be prokaryotic or eukaryotic. The term also includes anyprogeny of the subject host cell. It is understood that all progeny maynot be identical to the parental cell since there may be mutations thatoccur during replication. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart.

Hosts can include microbial, yeast, insect and mammalian organisms.Methods of expressing DNA sequences having eukaryotic or viral sequencesin prokaryotes are well known in the art. Non-limiting examples ofsuitable host cells include bacteria, archea, insect, fungi (forexample, yeast), plant, and animal cells (for example, mammalian cells,such as human). Exemplary cells of use include Escherichia coli,Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9cells, C129 cells, 293 cells, Neurospora, and immortalized mammalianmyeloid and lymphoid cell lines. Techniques for the propagation ofmammalian cells in culture are well-known (see, e.g., Helgason andMiller (Eds.), 2012, Basic Cell Culture Protocols (Methods in MolecularBiology), 4^(th) Ed., Humana Press). Examples of commonly used mammalianhost cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, andCOS cell lines, although cell lines may be used, such as cells designedto provide higher expression, desirable glycosylation patterns, or otherfeatures. In some embodiments, the host cells include HEK293 cells orderivatives thereof, such as GnTI^(−/−) cells (ATCC® No. CRL-3022), orHEK-293F cells.

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques. Where the host is prokaryotic, such as, but notlimited to, E. coli, competent cells which are capable of DNA uptake canbe prepared from cells harvested after exponential growth phase andsubsequently treated by the CaCl₂) method using standard procedures.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or viral vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding a disclosedantigen, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, ViralExpression Vectors, Springer press, Muzyczka ed., 2011). Appropriateexpression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

In one non-limiting example, a disclosed immunogen is expressed usingthe pVRC8400 vector (described in Barouch et al., J. Virol., 79,8828-8834, 2005, which is incorporated by reference herein).Modifications can be made to a nucleic acid encoding a disclosedrecombinant coronavirus S ectodomain protomer without diminishing itsbiological activity. Some modifications can be made to facilitate thecloning, expression, or incorporation of the targeting molecule into afusion protein. Such modifications are well known to those of skill inthe art and include, for example, termination codons, a methionine addedat the amino terminus to provide an initiation, site, additional aminoacids placed on either terminus to create conveniently locatedrestriction sites, or additional amino acids (such as poly His) to aidin purification steps.

In some embodiments, the disclosed recombinant coronavirus S ectodomainprotomer can be expressed in cells under conditions where therecombinant coronavirus S ectodomain protomer can self-assemble intotrimers which are secreted from the cells into the cell media. In suchembodiments, each recombinant coronavirus S ectodomain protomer containsa leader sequence (signal peptide) that causes the protein to enter thesecretory system, where the signal peptide is cleaved and the protomersform a trimer, before being secreted in the cell media. The medium canbe centrifuged and recombinant coronavirus S ectodomain trimer purifiedfrom the supernatant.

IV. Viral Vectors

A nucleic acid molecule encoding a protomer of a disclosed recombinantcoronavirus S ectodomain trimer can be included in a viral vector, forexample, for expression of the immunogen in a host cell, or forimmunization of a subject as disclosed herein. In some embodiments, theviral vectors are administered to a subject as part of a prime-boostvaccination. In several embodiments, the viral vectors are included in avaccine, such as a primer vaccine or a booster vaccine for use in aprime-boost vaccination.

In several examples, the viral vector can be replication-competent. Forexample, the viral vector can have a mutation in the viral genome thatdoes not inhibit viral replication in host cells. The viral vector alsocan be conditionally replication-competent. In other examples, the viralvector is replication-deficient in host cells.

A number of viral vectors have been constructed, that can be used toexpress the disclosed antigens, including polyoma, i.e., SV40 (Madzak etal., 1992, J. Gen. Virol., 73:15331536), adenovirus (Berkner, 1992, Cur.Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988, BioTechniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412;Quantin et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584;Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl.Acids Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. GeneTher., 1:241-256), vaccinia virus (Mackett et al., 1992, Biotechnology,24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top.Microbiol. Immunol., 158:91-123; On et al., 1990, Gene, 89:279-282),herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top.Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol.,66:29522965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield etal., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem.Pharmacol., 40:2189-2199), Sindbis viruses (H. Herweijer et al., 1995,Human Gene Therapy 6:1161-1167; U.S. Pat. Nos. 5,091,309 and5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol.11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA93:11371-11377) and retroviruses of avian (Brandyopadhyay et al., 1984,Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol.,66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol.,158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al.,1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol.,54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors are also known in the art, and may be obtained fromcommercial sources (such as PharMingen, San Diego, Calif.; ProteinSciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

In several embodiments, the viral vector can include an adenoviralvector that expresses a protomer of a disclosed recombinant coronavirusS ectodomain trimer. Adenovirus from various origins, subtypes, ormixture of subtypes can be used as the source of the viral genome forthe adenoviral vector. Non-human adenovirus (e.g., simian, chimpanzee,gorilla, avian, canine, ovine, or bovine adenoviruses) can be used togenerate the adenoviral vector. For example, a simian adenovirus can beused as the source of the viral genome of the adenoviral vector. Asimian adenovirus can be of serotype 1, 3, 7, 11, 16, 18, 19, 20, 27,33, 38, 39, 48, 49, 50, or any other simian adenoviral serotype. Asimian adenovirus can be referred to by using any suitable abbreviationknown in the art, such as, for example, SV, SAdV, SAV or sAV. In someexamples, a simian adenoviral vector is a simian adenoviral vector ofserotype 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, or 39. In one example, achimpanzee serotype C Ad3 vector is used (see, e.g., Peruzzi et al.,Vaccine, 27:1293-1300, 2009). Human adenovirus can be used as the sourceof the viral genome for the adenoviral vector. Human adenovirus can beof various subgroups or serotypes. For instance, an adenovirus can be ofsubgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2,5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22,23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36-39, and 42-48), subgroup E(e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), anunclassified serogroup (e.g., serotypes 49 and 51), or any otheradenoviral serotype. The person of ordinary skill in the art is familiarwith replication competent and deficient adenoviral vectors (includingsingly and multiply replication deficient adenoviral vectors). Examplesof replication-deficient adenoviral vectors, including multiplyreplication-deficient adenoviral vectors, are disclosed in U.S. Pat.Nos. 5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and7,195,896, and International Patent Application Nos. WO 94/28152, WO95/02697, WO 95/16772, WO 95/34671, WO 96/22378, WO 97/12986, WO97/21826, and WO 03/02231 1.

V. Virus-Like Particles

In some embodiments, a virus-like particle (VLP) is provided thatincludes a disclosed recombinant coronavirus S ectodomain trimer.Typically such VLPs include a recombinant coronavirus S ectodomaintrimer that is membrane anchored by a C-terminal transmembrane domain,for example the recombinant coronavirus S ectodomain protomers in thetrimer each can be linked to a transmembrane domain and cytosolic tailfrom the corresponding coronavirus. VLPs lack the viral components thatare required for virus replication and thus represent a highlyattenuated, replication-incompetent form of a virus. However, the VLPcan display a polypeptide (e.g., a recombinant coronavirus S ectodomaintrimer) that is analogous to that expressed on infectious virusparticles and can eliciting an immune response to the correspondingcoronavirus when administered to a subject. Virus like particles andmethods of their production are known and familiar to the person ofordinary skill in the art, and viral proteins from several viruses areknown to form VLPs, including human papillomavirus, HIV (Kang et al.,Biol. Chem. 380: 353-64 (1999)), Semliki-Forest virus (Notka et al.,Biol. Chem. 380: 341-52 (1999)), human polyomavirus (Goldmann et al., J.Virol. 73: 4465-9 (1999)), rotavirus (Jiang et al., Vaccine 17: 1005-13(1999)), parvovirus (Casal, Biotechnology and Applied Biochemistry, Vol29, Part 2, pp 141-150 (1999)), canine parvovirus (Hurtado et al., J.Virol. 70: 5422-9 (1996)), hepatitis E virus (Li et al., J. Virol. 71:7207-13 (1997)), and Newcastle disease virus. The formation of such VLPscan be detected by any suitable technique. Examples of suitabletechniques known in the art for detection of VLPs in a medium include,e.g., electron microscopy techniques, dynamic light scattering (DLS),selective chromatographic separation (e.g., ion exchange, hydrophobicinteraction, and/or size exclusion chromatographic separation of theVLPs) and density gradient centrifugation.

VI. Immunogenic Compositions

Immunogenic compositions comprising a disclosed immunogen (e.g., adisclosed recombinant coronavirus S ectodomain trimer or nucleic acidmolecule encoding a protomer of disclosed recombinant coronavirus Sectodomain trimer) and a pharmaceutically acceptable carrier are alsoprovided. Such pharmaceutical compositions can be administered tosubjects by a variety of administration modes known to the person ofordinary skill in the art, for example, intramuscular, intradermal,subcutaneous, intravenous, intra-arterial, intra-articular,intraperitoneal, intranasal, sublingual, tonsillar, oropharyngeal, orother parenteral and mucosal routes. In several embodiments,pharmaceutical compositions including one or more of the disclosedimmunogens are immunogenic compositions. Actual methods for preparingadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemingtons Pharmaceutical Sciences, 19^(th) Ed., Mack PublishingCompany, Easton, Pa., 1995.

Thus, an immunogen described herein can be formulated withpharmaceutically acceptable carriers to help retain biological activitywhile also promoting increased stability during storage within anacceptable temperature range. Potential carriers include, but are notlimited to, physiologically balanced culture medium, phosphate buffersaline solution, water, emulsions (e.g., oil/water or water/oilemulsions), various types of wetting agents, cryoprotective additives orstabilizers such as proteins, peptides or hydrolysates (e.g., albumin,gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g.,sodium glutamate), or other protective agents. The resulting aqueoussolutions may be packaged for use as is or lyophilized. Lyophilizedpreparations are combined with a sterile solution prior toadministration for either single or multiple dosing.

Formulated compositions, especially liquid formulations, may contain abacteriostat to prevent or minimize degradation during storage,including but not limited to effective concentrations (usually 1% w/v)of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben,and/or propylparaben. A bacteriostat may be contraindicated for somepatients; therefore, a lyophilized formulation may be reconstituted in asolution either containing or not containing such a component.

The immunogenic compositions of the disclosure can contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate. The immunogenic composition may optionally include an adjuvantto enhance an immune response of the host. Suitable adjuvants are, forexample, toll-like receptor agonists, alum, AlPO4, alhydrogel, Lipid-Aand derivatives or variants thereof, oil-emulsions, saponins, neutralliposomes, liposomes containing the vaccine and cytokines, non-ionicblock copolymers, and chemokines. Non-ionic block polymers containingpolyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POEblock copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa,Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), amongmany other suitable adjuvants well known in the art, may be used as anadjuvant (Newman et al., 1998, Critical Reviews in Therapeutic DrugCarrier Systems 15:89-142). These adjuvants have the advantage in thatthey help to stimulate the immune system in a non-specific way, thusenhancing the immune response to a pharmaceutical product.

In some instances it may be desirable to combine a disclosed immunogen,with other pharmaceutical products (e.g., vaccines) which induceprotective responses to other agents. For example, a compositionincluding a recombinant paramyxovirus as described herein can be can beadministered simultaneously (typically separately) or sequentially withother vaccines recommended by the Advisory Committee on ImmunizationPractices (ACIP; cdc.gov/vaccines/acip/index.html) for the targeted agegroup (e.g., infants from approximately one to six months of age), suchas an influenza vaccine or a varicella zoster vaccine. As such, adisclosed immunogen including a recombinant coronavirus S ectodomaintrimer described herein may be administered simultaneously orsequentially with vaccines against, for example, hepatitis B (HepB),diphtheria, tetanus and pertussis (DTaP), pneumococcal bacteria (PCV),Haemophilus influenzae type b (Hib), polio, influenza and rotavirus.

In some embodiments, the composition can be provided as a sterilecomposition. The pharmaceutical composition typically contains aneffective amount of a disclosed immunogen and can be prepared byconventional techniques. Typically, the amount of immunogen in each doseof the immunogenic composition is selected as an amount which induces animmune response without significant, adverse side effects. In someembodiments, the composition can be provided in unit dosage form for useto induce an immune response in a subject. A unit dosage form contains asuitable single preselected dosage for administration to a subject, orsuitable marked or measured multiples of two or more preselected unitdosages, and/or a metering mechanism for administering the unit dose ormultiples thereof. In other embodiments, the composition furtherincludes an adjuvant.

VII. Methods of Inducing an Immune Response

The disclosed immunogens (e.g., recombinant coronavirus S ectodomaintrimer, a nucleic acid molecule (such as an RNA molecule) or vectorencoding a protomer of a disclosed recombinant coronavirus S ectodomaintrimer, or a protein nanoparticle or virus like particle comprising adisclosed recombinant coronavirus S ectodomain trimer) can beadministered to a subject to induce an immune response to thecorresponding coronavirus S ectodomain in the subject. In a particularexample, the subject is a human. The immune response can be a protectiveimmune response, for example a response that inhibits subsequentinfection with the corresponding coronavirus. Elicitation of the immuneresponse can also be used to treat or inhibit infection and illnessesassociated with the corresponding coronavirus.

A subject can be selected for treatment that has, or is at risk fordeveloping infection with the coronavirus corresponding to the S proteinin the immunogen, for example because of exposure or the possibility ofexposure to the coronavirus. Following administration of a disclosedimmunogen, the subject can be monitored for infection or symptomsassociated with the coronavirus, or both.

Typical subjects intended for treatment with the therapeutics andmethods of the present disclosure include humans, as well as non-humanprimates and other animals. To identify subjects for prophylaxis ortreatment according to the methods of the disclosure, accepted screeningmethods are employed to determine risk factors associated with atargeted or suspected disease or condition, or to determine the statusof an existing disease or condition in a subject. These screeningmethods include, for example, conventional work-ups to determineenvironmental, familial, occupational, and other such risk factors thatmay be associated with the targeted or suspected disease or condition,as well as diagnostic methods, such as various ELISA and otherimmunoassay methods to detect and/or characterize coronavirus infection.These and other routine methods allow the clinician to select patientsin need of therapy using the methods and pharmaceutical compositions ofthe disclosure. In accordance with these methods and principles, acomposition can be administered according to the teachings herein, orother conventional methods, as an independent prophylaxis or treatmentprogram, or as a follow-up, adjunct or coordinate treatment regimen toother treatments.

The administration of a disclosed immunogen can be for prophylactic ortherapeutic purpose. When provided prophylactically, the disclosedtherapeutic agents are provided in advance of any symptom, for example,in advance of infection. The prophylactic administration of thedisclosed therapeutic agents serves to prevent or ameliorate anysubsequent infection. When provided therapeutically, the disclosedtherapeutic agents are provided at or after the onset of a symptom ofdisease or infection, for example, after development of a symptom ofinfection with the coronavirus corresponding to the S protein in theimmunogen, or after diagnosis with the coronavirus infection. Thetherapeutic agents can thus be provided prior to the anticipatedexposure to the coronavirus so as to attenuate the anticipated severity,duration or extent of an infection and/or associated disease symptoms,after exposure or suspected exposure to the virus, or after the actualinitiation of an infection.

The immunogens described herein, and immunogenic compositions thereof,are provided to a subject in an amount effective to induce or enhance animmune response against the coronavirus S protein in the immunogen inthe subject, preferably a human. The actual dosage of disclosedimmunogen will vary according to factors such as the disease indicationand particular status of the subject (for example, the subject's age,size, fitness, extent of symptoms, susceptibility factors, and thelike), time and route of administration, other drugs or treatments beingadministered concurrently, as well as the specific pharmacology of thecomposition for eliciting the desired activity or biological response inthe subject. Dosage regimens can be adjusted to provide an optimumprophylactic or therapeutic response.

An immunogenic composition including one or more of the disclosedimmunogens can be used in coordinate (or prime-boost) vaccinationprotocols or combinatorial formulations. In certain embodiments, novelcombinatorial immunogenic compositions and coordinate immunizationprotocols employ separate immunogens or formulations, each directedtoward eliciting an anti-viral immune response, such as an immuneresponse to coronavirus S proteins. Separate immunogenic compositionsthat elicit the anti-viral immune response can be combined in apolyvalent immunogenic composition administered to a subject in a singleimmunization step, or they can be administered separately (in monovalentimmunogenic compositions) in a coordinate (or prime-boost) immunizationprotocol.

There can be several boosts, and each boost can be a different disclosedimmunogen. In some examples that the boost may be the same immunogen asanother boost, or the prime. The prime and boost can be administered asa single dose or multiple doses, for example two doses, three doses,four doses, five doses, six doses or more can be administered to asubject over days, weeks or months. Multiple boosts can also be given,such one to five (e.g., 1, 2, 3, 4 or 5 boosts), or more. Differentdosages can be used in a series of sequential immunizations. For examplea relatively large dose in a primary immunization and then a boost withrelatively smaller doses.

In some embodiments, the boost can be administered about two, aboutthree to eight, or about four, weeks following the prime, or aboutseveral months after the prime. In some embodiments, the boost can beadministered about 5, about 6, about 7, about 8, about 10, about 12,about 18, about 24, months after the prime, or more or less time afterthe prime. Periodic additional boosts can also be used at appropriatetime points to enhance the subject's “immune memory.” The adequacy ofthe vaccination parameters chosen, e.g., formulation, dose, regimen andthe like, can be determined by taking aliquots of serum from the subjectand assaying antibody titers during the course of the immunizationprogram. In addition, the clinical condition of the subject can bemonitored for the desired effect, e.g., prevention of infection orimprovement in disease state (e.g., reduction in viral load). If suchmonitoring indicates that vaccination is sub-optimal, the subject can beboosted with an additional dose of immunogenic composition, and thevaccination parameters can be modified in a fashion expected topotentiate the immune response.

In some embodiments, the prime-boost method can include DNA-primer andprotein-boost vaccination protocol to a subject. The method can includetwo or more administrations of the nucleic acid molecule or the protein.

For protein therapeutics, typically, each human dose will comprise1-1000 μg of protein, such as from about 1 μg to about 100 μg, forexample, from about 1 μg to about 50 μg, such as about 1 μg, about 2 μg,about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30μg, about 40 μg, or about 50 μg.

The amount utilized in an immunogenic composition is selected based onthe subject population (e.g., infant or elderly). An optimal amount fora particular composition can be ascertained by standard studiesinvolving observation of antibody titers and other responses insubjects. It is understood that a therapeutically effective amount of adisclosed immunogen, such as a disclosed recombinant coronavirus Sectodomain trimer, viral vector, or nucleic acid molecule in aimmunogenic composition, can include an amount that is ineffective ateliciting an immune response by administration of a single dose, butthat is effective upon administration of multiple dosages, for examplein a prime-boost administration protocol.

Upon administration of a disclosed immunogen of this disclosure, theimmune system of the subject typically responds to the immunogeniccomposition by producing antibodies specific for the coronavirus Sectodomain trimer included in the immunogen. Such a response signifiesthat an immunologically effective dose was delivered to the subject.

In some embodiments, the antibody response of a subject will bedetermined in the context of evaluating effective dosages/immunizationprotocols. In most instances it will be sufficient to assess theantibody titer in serum or plasma obtained from the subject. Decisionsas to whether to administer booster inoculations and/or to change theamount of the therapeutic agent administered to the individual can be atleast partially based on the antibody titer level. The antibody titerlevel can be based on, for example, an immunobinding assay whichmeasures the concentration of antibodies in the serum which bind to anantigen including, for example, the recombinant coronavirus S ectodomaintrimer included in the immunogen.

Coronavirus infection does not need to be completely eliminated orreduced or prevented for the methods to be effective. For example,elicitation of an immune response to a coronavirus with one or more ofthe disclosed immunogens can reduce or inhibit infection with thecoronavirus by a desired amount, for example, by at least 10%, at least20%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, or even at least 100% (elimination orprevention of detectable infected cells), as compared to infection withthe coronavirus in the absence of the immunogen. In additional examples,coronavirus replication can be reduced or inhibited by the disclosedmethods. Coronavirus replication does not need to be completelyeliminated for the method to be effective. For example, the immuneresponse elicited using one or more of the disclosed immunogens canreduce replication of the corresponding coronavirus by a desired amount,for example, by at least 10%, at least 20%, at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, oreven at least 100% (elimination or prevention of detectable replicationof the coronavirus), as compared to replication of the coronavirus inthe absence of the immune response.

In some embodiments, the disclosed immunogen is administered to thesubject simultaneously with the administration of the adjuvant. In otherembodiments, the disclosed immunogen is administered to the subjectafter the administration of the adjuvant and within a sufficient amountof time to induce the immune response.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid.Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. Nos.5,593,972 and 5,817,637 (which describe operably linking a nucleic acidsequence encoding an antigen to regulatory sequences enablingexpression). U.S. Pat. No. 5,880,103 describes several methods ofdelivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™, negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A™ (saponin). Protective immunity has beengenerated in a variety of experimental models of infection, includingtoxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ asthe delivery vehicle for antigens (Mowat and Donachie, Immunol. Today12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™have been found to produce Class I mediated CTL responses (Takahashi etal., Nature 344:873, 1990).

In some embodiments, a plasmid DNA vaccine is used to express adisclosed immunogen in a subject. For example, a nucleic acid moleculeencoding a disclosed immunogen can be administered to a subject toinduce an immune response to the coronavirus S protein included in theimmunogen. In some embodiments, the nucleic acid molecule can beincluded on a plasmid vector for DNA immunization, such as the pVRC8400vector (described in Barouch et al., J. Virol, 79, 8828-8834, 2005,which is incorporated by reference herein).

In another approach to using nucleic acids for immunization, a disclosedrecombinant coronavirus S ectodomain or recombinant coronavirus Sectodomain trimer can be expressed by attenuated viral hosts or vectorsor bacterial vectors. Recombinant vaccinia virus, adeno-associated virus(AAV), herpes virus, retrovirus, cytogmeglo virus or other viral vectorscan be used to express the peptide or protein, thereby eliciting a CTLresponse. For example, vaccinia vectors and methods useful inimmunization protocols are described in U.S. Pat. No. 4,722,848. BCG(Bacillus Calmette Guerin) provides another vector for expression of thepeptides (see Stover, Nature 351:456-460, 1991).

In one embodiment, a nucleic acid encoding a disclosed recombinantcoronavirus S ectodomain or coronavirus S ectodomain trimer isintroduced directly into cells. For example, the nucleic acid can beloaded onto gold microspheres by standard methods and introduced intothe skin by a device such as Bio-Rad's HELIOS™ Gene Gun. The nucleicacids can be “naked,” consisting of plasmids under control of a strongpromoter. Typically, the DNA is injected into muscle, although it canalso be injected directly into other sites. Dosages for injection areusually around 0.5 μg/kg to about 50 mg/kg, and typically are about0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Pat. No. 5,589,466).

For example, the nucleic acid can be loaded onto gold microspheres bystandard methods and introduced into the skin by a device such asBio-Rad's HELIOS™ Gene Gun. The nucleic acids can be “naked,” consistingof plasmids under control of a strong promoter. Typically, the DNA isinjected into muscle, although it can also be injected directly intoother sites. Dosages for injection are usually around 0.5 μg/kg to about50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see,e.g., U.S. Pat. No. 5,589,466).

In another embodiment, an mRNA-based immunization protocol can be usedto deliver a nucleic acid encoding a disclosed recombinant coronavirus Sectodomain or coronavirus S ectodomain trimer directly into cells. Insome embodiments, nucleic acid-based vaccines based on mRNA may providea potent alternative to the previously mentioned approaches. mRNAvaccines preclude safety concerns about DNA integration into the hostgenome and can be directly translated in the host cell cytoplasm.Moreover, the simple cell-free, in vitro synthesis of RNA avoids themanufacturing complications associated with viral vectors. Two exemplaryforms of RNA-based vaccination that can be used to deliver a nucleicacid encoding a disclosed recombinant coronavirus S ectodomain orcoronavirus S ectodomain trimer include conventional non-amplifying mRNAimmunization (see, e.g., Petsch et al., “Protective efficacy of in vitrosynthesized, specific mRNA vaccines against influenza A virusinfection,” Nature biotechnology, 30(12):1210-6, 2012) andself-amplifying mRNA immunization (see, e.g., Geall et al., “Nonviraldelivery of self-amplifying RNA vaccines,” PNAS, 109(36): 14604-14609,2012; Magini et al., “Self-Amplifying mRNA Vaccines Expressing MultipleConserved Influenza Antigens Confer Protection against Homologous andHeterosubtypic Viral Challenge,” PLoS One, 11(8):e0161193, 2016; andBrito et al., “Self-amplifying mRNA vaccines,” Adv Genet., 89:179-233,2015).

In some embodiments, administration of a therapeutically effectiveamount of one or more of the disclosed immunogens to a subject induces aneutralizing immune response in the subject. To assess neutralizationactivity, following immunization of a subject, serum can be collectedfrom the subject at appropriate time points, frozen, and stored forneutralization testing. Methods to assay for neutralization activity areknown to the person of ordinary skill in the art and are furtherdescribed herein, and include, but are not limited to, plaque reductionneutralization (PRNT) assays, microneutralization assays, flow cytometrybased assays, single-cycle infection assays. In some embodiments, theserum neutralization activity can be assayed using a panel ofcoronavirus pseudoviruses. For example, to test the immunogenicity ofthe vaccine candidates against multiple MERS-CoV strains—without therequirement of a biosafety level 3 facility—a pseudotyped reporter virusneutralization assay was previously developed (Wand et al., Nat Commun,6:7712, 2015), similar to that previously developed for SARS-CoV (Martinet al., Vaccine 26, 6338, 2008; Yang et al., Nature 428, 561, 2004;Naldini et al., PNAS 93, 11382, 1996; Yang et al., PNAS 102, 797, 2005).

In some embodiments, administration of a therapeutically effectiveamount of one or more of the disclosed immunogens to a subject induces aneutralizing immune response in the subject. To assess neutralizationactivity, following immunization of a subject, serum can be collectedfrom the subject at appropriate time points, frozen, and stored forneutralization testing. Methods to assay for neutralization activity areknown to the person of ordinary skill in the art and are furtherdescribed herein, and include, but are not limited to, plaque reductionneutralization (PRNT) assays, microneutralization assays, flow cytometrybased assays, single-cycle infection assays. In some embodiments, theserum neutralization activity can be assayed using a panel ofcoronavirus pseudoviruses.

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Prefusion Stabilized MERS-CoV S Protein

This example describes development of a recombinant MERS-CoV Sectodomain trimer that is stabilized in a prefusion conformation.

The structure of the prefusion S ectodomain trimer of a humanbetacoronavirus was recently elucidated (Kirchdoerfer et al., “Prefusionstructure of a human coronavirus spike protein,” Nature, 351:118-121,2016). This structure was further investigated to reveal several keydetails about human coronavirus spike architecture. First,receptor-binding elements within S1 cap the fusion-mediating elements inS2, likely preventing their conformational rearrangement (FIG. 1) untiltriggering occurs.

The S1 C-terminal domains appear interdigitated and form extensivequaternary interactions (FIG. 2A), suggesting conformational flexibilityor “breathing” would be required for the HKU1-CoV Spike to make receptorinteractions similar to those made between the SARS-CoV receptor bindingdomain (RBD) and ACE2 receptors (FIG. 2A). In addition, the structurerevealed two sub-domains, SD-1 and SD-2, in HKU1-CoV S1 (FIGS. 2C, 2D).The SD-2 loop contains the site dedicated to HKU1-CoV S furin cleavage;and furin cleavage at the S1/S2 junction is a process necessary forinfection (FIG. 2D). S2 contains four classical components of a Class 1fusion machine: a fusion peptide (FIG. 3A), two heptad repeats, HR1(FIG. 3B) and HR2, and a transmembrane domain.

Structure-based stabilization of betacoronavirus prefusion trimers. TheHKU1-CoV prefusion S structure was used as a starting point to designmutations that would stabilize betacoronavirus S trimers in theprefusion conformation. Dozens of possible stabilizing mutations weredesigned and tested in the context of the MERS-CoV S protein. Twomutations were identified to be particularly effective for stabilizingthe MERS-CoV S protein in its prefusion conformation: V1060P and L1061P(or their combination) (FIG. 4). MERS-CoV S proteins including thesemutations also had >50 fold increased expression (FIG. 4). These twoproline substitutions are located at the top portion (membrane distal)of the MERS-CoV S2 central helix and HR1 to prevent pre-to-postfusionconformational changes. Prefusion stabilization of the MERS-CoV Sprotein is preliminarily indicated by increased expression levels whenthese mutations are combined compared to an S2 truncated, but otherwisewild-type (WT) MERS-CoV S (C6) (FIGS. 4B,4C). WT MERS-CoV S likelyspontaneously flips from pre-to-postfusion conformation. Correspondingdouble proline mutations in SARS-CoV and HKU1-CoV S also increasedexpression above WT S.

S protein immunogens were expressed from codon-optimized genes encodingthe S ectodomain (without TM and CT) with a C-terminal T4 fibritintrimerization domain, an HRV3C cleavage site, a 6×His-tag and aTwin-Strep-tag that were cloned into the eukaryotic-expression vectorpaH. Following sequence verification, expression plasmids weretransiently transfected into FreeStyle293 cells. Three hours aftertransfection, kifunensine was added to a final concentration of 5 μM.Cultures were harvested six days later, and secreted protein waspurified from the supernatant and soluble protein w purified from thesupernatant by passage over Ni²⁺-NTA and StrepTactin resin usingaffinity tags on the C-terminus of the proteins. The purified proteinswere then be passed over a size-exclusion column to assess theiroligomeric state and to isolate monodisperse fractions corresponding totrimeric ectodomains. Protein expression levels were then assessed bySDS-PAGE (10 μL of protein-bound resin was boiled and loaded per lane).This expression strategy was used to generated generate and testproline-substituted variants of MERS-CoV S (Eng1 strain, residues1-1291), SARS-CoV S (Tort strain, residues 1-1190) and HCoV-HKU1 S (N5strain, residues 1-1276). The MERS-CoV S ectodomain trimers included a748-RSVR-751 (residues 748-751 of SEQ ID NO: 1) to 748-ASVG-751(residues 748-751 of SEQ ID NO: 3) substitutions to remove the S1/S2cleavage site.

Mice (N=5/group) were vaccinated with 0.1 μg, 1 μg, or 10 μg of theMERS-CoV S trimer stabilized in the prefusion conformation by V1060P andL1061P substitutions to evaluate the effectiveness of the resultingimmune response (FIG. 5). As a comparison, mice were also vaccinatedwith the MERS-CoV S1 protein, which was previously found to inducerobust neutralizing antibody responses associated with protection, andMERS-CoV S ectodomain trimers with WT sequence. Control mice were givenPBS. The immunogens were based on the England1 (“Eng”) MERS-CoV strain.

Immunizations were performed as weeks 0 and 3. Two weeks following thelast immunization, serum was collected and tested for neutralizationagainst various MERS pseudovirus strains: England1, Florida USA2,Bisha1, Korea002, JordanN3, Buraidah1, and Indiana USA1. Serum wasdiluted, in triplicate, and incubated with MERS-CoV pseudovirus prior toinoculation of Huh7.5 cells. Dilution curves were fitted to mock cellsand cells exposed to un-neutralized virus as 100% and 0% neutralization,respectively. IC90 titers were calculated as the dilution of serumneeded to neutralize 90% of MERS-CoV pseudovirus.

Vaccination with the MERS-CoV S1, wild-type MERS-CoV S ectodomain, orthe prefusion stabilized MERS-CoV S ectodomain induced similar robustlevels of neutralizing antibodies against homologous MERS-CoV England1reporter pseudovirus at dosages of 10 μg, but the prefusion-stabilizedspike was superior at lower dosages (FIG. 5A). Further, when testedagainst homologous virus strains, the prefusion stabilized MERS-CoVectodomain trimer produced a superior immune response (FIG. 5B).

Additional assays were performed to show that vaccination with MERS S-2Pectodomain trimer elicited more non-RBD binding antibodies than MERS S1ectodomain trimer (FIG. 6A), and higher levels of neutralizing activitytargeting a greater diversity of epitopes than antigens based on RBD orS1 monomer (FIG. 6B).

Additionally, challenge studies were performed to determine if theprefusion-stabilized MERS-CoV S ectodomain trimer could prevent MERS-CoVinfection in an animal model (FIG. 7). The challenge studies wereperformed using C57BL/6J mice that were genetically engineered usingCRISPR-Cas9 genomic editing to encode human DPP4 mutations (288L and330R; “288/330^(+/+)”) as previously described (see, Cockrell et al., “Amouse model for MERS coronavirus-induced acute respiratory distresssyndrome.” Nature Microbiology. 2:16226, 2016, which is incorporated byreference herein). These mice are known to be susceptible to inventionwith MERS-CoV. The 288/330^(+/+) mice were vaccinated with 0.1 μg MERSCoV-S ectodomain timer with the double proline mutation using the SigmaAdjuvant System at weeks 0 and 3. Four weeks following finalvaccination, the mice were challenged with a lethal dose ofmouse-adapted MERS virus and monitored for survival and weight loss. Asshown in FIG. 7, prior immunization with the prefusion stabilizedMERS-CoV S ectodomain trimer protected against lethal MERS challenge inmice.

Example 2 Prefusion Stabilized Coronavirus Spike Proteins

HKU1-CoV is closely related to other betacoronaviruses, such as thezoonotic viruses SARS-CoV and MERS-CoV, both of which are associatedwith high mortality. Accordingly, additional coronavirus S ectodomaintrimers stabilized in the prefusion conformation by double prolinemutations at the HR1/central helix junction were evaluated as vaccinecandidates.

Due to the structural similarity of coronavirus S proteins, thesequences of these proteins can be readily aligned to identifystructural domains, such as the HR1 and central helix. FIG. 8illustrates the structural domains of the HKU1, SARS, and MERS-CoV Sproteins, as well as positioning of double proline substitutions tostabilize these proteins in the prefusion conformation. FIG. 8 shows asequence alignment of the S2 subunit of the HKU1-CoV, SARS-CoV,MERS-CoV, HKU9-CoV, NL63-CoV, and 229E-CoV S proteins, showing relevantsequence homology. The HR1 spans the α13, α14, α17, and α16 helixes,including approximately residues 996-1064 (relative to HKU1-CoVnumbering shown in the figure). The central helix is the α17 helix,including approximately residues 1068-1110. The HR2 includesapproximates residues 1245-1276 (relative to HKU1-CoV numbering shown inthe figure). The transmembrane domain begins at approximately residue1292 (relative to HKU1-CoV numbering shown in the figure).

Proline substitutions were introduced into the SARS-CoV, HKU1-CoV,OC43-CoV, HKU9-CoV, WIV1-CoV, MHV-CoV, NL63-CoV and 229E-CoV. TheSARS-CoV substitutions were K968P, V969P, or K968P and V969P. TheHKU1-CoV substitutions are N1067P, L1068P, or N1067P and L1068P. TheOC43-CoV substitutions are A1079P, L1080P, or A1079P and L1080P. TheHKU9-CoV substitutions are G983P, L984P, or G983P and L984P. TheWIV1-CoV substitutions are K969P, V970P, or K969P and V970P. The MHV-CoVsubstitutions are A1073P, L1074P, or A1073P and L1074P. The NL63-CoVsubstitutions are S1052P, I1053P, or S1052P and I1053P. The 229E-CoVsubstitutions are I869P, I870P, or I869P and I870P. Soluble SARS-CoV,HKU1-CoV, OC43-CoV, HKU9-CoV, WIV1-CoV, MHV-CoV, NL63-CoV and 229E-CoV Sectodomain trimers containing the indicated mutations, a signal peptide,and a C-terminal linkage to a T4 Fibritin trimerization domain andstreptavidin tag were expressed in cells and purified as described inExample 1. Including the signal peptide and T4 Fibritin trimerizationdomain, protomer sequences of the referenced ectodomain trimersincluding the double proline substitutions are as follows:

SARS-CoV S 2P (K968P and V969P, SEQ ID NO: 30)

HKU1-CoV S 2P (N1067P and L1068P, SEQ ID NO: 31)

HKU9-CoV S 2P (G983P and L984P, SEQ ID NO: 32)

OC43-CoV S 2P (A1079P and L1080P, SEQ ID NO: 33)

WIV1-CoV S 2P (K969P and V970P, SEQ ID NO: 34)

MHV-CoV S 2P (A1073P and L1074P, SEQ ID NO: 35)

NL63-CoV S 2P (51052P and I1053P, SEQ ID NO: 36)

229E-CoV S 2P (I869P and I870P, SEQ ID NO: 37)

PEDV-CoV S 2P (I1076P and L1077P, SEQ ID NO: 40)

As shown in FIG. 10, the proline substitutions boosted the expression ofthe SARS-CoV and HKU1-CoV S ectodomains.

The thermal stability of the wild-type SARS-CoV S ectodomain (SARS-S-WT)and SARS-CoV S ectodomain with K968P and V969P (SARS-S-2P) was assessed(FIG. 11). About 3 μg SARS-S-WT or SARS-S-2P samples in TBS buffer (2 mMTris pH8.0, 200 mM NaCl) were incubated at different temperature for 1hour. The samples were then analyzed on the NativePAGE Novex Bis-Trisgels (Invitrogen) using procedures suggested by the manufacturer. Asshown in FIG. 11, the SARS-S-2P has higher thermal stability thanSARS-S-WT.

The expressed protein trimers were further analyzed by gelchromatography. FIG. 12 illustrates results from chromatographyexperiments concerning wild-type SARS-CoV S ectodomain (SARS-S-WT),SARS-CoV S ectodomain with K968P and V969P (SARS-S-2P), wild-typeMERS-CoV S ectodomain (MERS-S-WT), MERS-CoV S ectodomain with V1060P andL1061P (MERS-S-2P), wild-type HKU1-CoV S ectodomain (HKU1-S-WT),HKU1-CoV S ectodomain with N1067P and L1068P (HKU1-S-2P). In all threecases, a larger peak was observed for the double proline mutant, show amany-fold increase in expression of the double proline mutant relativeto the WT ectodomain trimer.

The conformation of the double proline mutant SARS-CoV, HKU1-CoV, andMERS-CoV S variants was assessed by negative stain electron microscopy(FIG. 13A). In each case the S variants with the double proline mutantwere homogeneous and form trimers in the expected prefusion shape. Eachof these ectodomain trimers was purified as a single peak and formedtrimers in the typical prefusion conformation. In contrast,corresponding S proteins with native sequences formed trimers of mixedconformation, with some trimers in the typical prefusion conformationand others in the typical elongated post-fusion conformation.

Additionally, the conformation of the double proline mutant OC43-CoV,WIV1-CoV, and PEDV-CoV, and 229E-CoV S variants was also assessed bynegative stain electron microscopy (FIGS. 13B-13C). In each case the Svariants with the double proline mutant were homogeneous and formtrimers in the expected prefusion shape. Each of these ectodomaintrimers was purified as a single peak and formed trimers in the typicalprefusion conformation.

When low resolution negative stain reconstructions of S trimerconstructs from HKU1-CoV (FIG. 14A), MERS-CoV (FIG. 14B), SARS-CoV (FIG.14C), OC43-CoV S 2P (FIG. 14D), WIV1-CoV S 2P (FIG. 14E), PEDV-CoV S 2P(FIG. 14F), and 229E S-2P (FIG. 14G) were reconstructed from the EMdata, the articles all formed homogeneous trimeric spike proteinstructures.

To assess the immunogenicity of the SARS-CoV S 2P ectodomain trimer,mice (N=5/group) were vaccinated with 0.1 μg or 1 μg of the SARS-CoV Strimer stabilized in the prefusion conformation by K968P and V969Psubstitutions (SEQ ID NO: 30) to evaluate the effectiveness of theresulting immune response (FIG. 15). As a comparison, mice were alsovaccinated with the SARS-CoV S ectodomain trimers with WT sequence. Theimmunogens were based on the TOR2 SARS-CoV strain. Immunizations wereperformed as weeks 0 and 3. Two weeks following the last immunization,serum was collected and tested for neutralization against autologousSARS pseudovirus. Serum was diluted, in triplicate, and incubated withSARS-CoV pseudovirus prior to inoculation of Huh7.5 cells. Dilutioncurves were fitted to mock cells and cells exposed to un-neutralizedvirus as 100% and 0% neutralization, respectively. IC90 titers werecalculated as the dilution of serum needed to neutralize 90% of SARS-CoVpseudovirus. As shown in FIG. 15, vaccination with the prefusionstabilized SARS-CoV S ectodomain induced a superior immune responserelative to the wild-type SARS-CoV S ectodomain, particularly at the 0.1μg dose.

Additionally, mice (N=5/group) were vaccinated with 0.1 μg, 1 μg, or 10μg of the HKU1-CoV S trimer stabilized in the prefusion conformation byN1067P and L1068P substitutions (SEQ ID NO: 31) to evaluate theeffectiveness of the resulting immune response (FIG. 15). As acomparison, mice were also vaccinated with the HKU1-CoV S ectodomaintrimers with WT sequence. Immunizations were performed as weeks 0 and 3.Two weeks following the last immunization, serum was collected andtested for neutralization against autologous HKU1-CoV pseudovirus. Serumwas diluted, in triplicate, and incubated with HKU1-CoV pseudovirusprior to inoculation of Huh7.5 cells. Dilution curves were fitted tomock cells and cells exposed to un-neutralized virus as 100% and 0%neutralization, respectively. IC90 titers were calculated as thedilution of serum needed to neutralize 90% of HKU1-CoV pseudovirus. Asshown in FIG. 15, vaccination with the prefusion stabilized HKU1-CoV Sectodomain induced a superior immune response relative to the wild-typeHKU1-CoV S ectodomain, particularly at the 0.1 μg dose.

In additional assays, mice (N=5/group) were vaccinated with 1 μg of theOC43-CoV S ectodomain trimer stabilized in the prefusion conformation byA1079P and L1080P substitutions (SEQ ID NO: 33) or with 1 μg of theWIV1-CoV S ectodomain trimer stabilized in the prefusion conformation byK969P and V970P substitutions (SEQ ID NO: 34) to evaluate theeffectiveness of the resulting immune response (FIG. 16). PBS was usedas a control. Immunizations were performed as weeks 0 and 3. Two weeksfollowing the last immunization, serum was collected and tested forbinding to the corresponding immunogen by ELISA. As shown in FIG. 16,vaccination with the prefusion stabilized OC43-CoV S ectodomain trimeror the prefusion stabilized WIV1-CoV S ectodomain trimer elicitedantibodies that target the corresponding ectodomain trimers.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. An immunogen, comprising: a recombinant coronavirus S ectodomain trimer comprising protomers comprising one or two proline substitutions at or near a junction between a heptad repeat 1 (HR1) and a central helix that stabilize the S ectodomain trimer in a prefusion conformation.
 2. The immunogen of claim 1, wherein the one or two proline substitutions comprise two consecutive proline substitutions.
 3. The immunogen of claim 1, wherein the one or two proline substitutions are located between a position 15 amino acids N-terminal of a C-terminal residue of the HR1 and a position 5 amino acids C-terminal of an N-terminal residue of the central helix.
 4. The immunogen of claim 1, wherein the coronavirus is one of MERS-CoV, SARS-CoV, NL63-CoV, 229E-CoV, OC43-CoV, HKU1-CoV, WIV1-CoV, MHV, HKU9-CoV, PEDV-CoV, or SDCV.
 5. The immunogen of claim 1, wherein the coronavirus is a betacoronavirus.
 6. The immunogen of claim 1, wherein the recombinant coronavirus S ectodomain trimer is a recombinant MERS-CoV S ectodomain trimer, and the one or two proline substitutions are between residues 1050 to 1069 of the protomers in the trimer.
 7. The immunogen of claim 6, wherein the one or two proline substitutions are between residues 1053 to 1063 of the protomers in the trimer.
 8. The immunogen of claim 6, wherein the one or two proline substitutions are between residues 1058 to 1063 of the protomers in the trimer.
 9. The immunogen of claim 6, wherein the one or two proline substitutions are V1060P and/or L1061P substitutions.
 10. The immunogen of claim 6, wherein the protomers in the recombinant MERS-CoV S ectodomain trimer comprise the amino acid sequence set forth as the ectodomain of any one of SEQ ID NOs: 2-4, or an amino acid sequence at least 90% identical thereto wherein the protomers comprise the one or two proline substitutions for stabilization in the ectodomain trimer in the prefusion conformation.
 11. The immunogen of claim 10, wherein the protomers in the recombinant MERS-CoV S ectodomain trimer comprise residues 18-1291 of any one of SEQ ID NOs: 2-4, or an amino acid sequence at least 90% identical thereto wherein the protomers comprise the one or two proline substitutions for stabilization in the ectodomain trimer in the prefusion conformation.
 12. The immunogen of claim 6, wherein a C-terminal residue of the protomers in the ectodomain is linked to a trimerization domain by a peptide linker, or is directly linked to the trimerization domain.
 13. The immunogen of claim 12, wherein the trimerization domain is a T4 fibritin trimerization domain.
 14. The immunogen of claim 13, wherein the protomers linked to the T4 fibritin trimerization domain in the recombinant coronavirus S ectodomain trimer comprise residues 18-1318 of SEQ ID NO: 29 (MERS-2P), or an amino acid sequence at least 90% identical to any one of residues 18-1318 of SEQ ID NO: 29 (MERS-2P), wherein the protomers comprise the one or two proline substitutions for stabilization in the ectodomain trimer in the prefusion conformation.
 15. The immunogen of claim 1, wherein the recombinant coronavirus S ectodomain trimer is a recombinant SARS-CoV S ectodomain trimer, and the one or two proline substitutions are between residues 951 to 971 of the protomers in the trimer.
 16. The immunogen of claim 15, wherein the one or two proline substitutions are between residues 961 to 971 of the protomers in the trimer.
 17. The immunogen of claim 15, wherein the one or two proline substitutions are between residues 966 to 971 of the protomers in the trimer.
 18. The immunogen of claim 15, wherein the one or two proline substitutions are K968P and/or V969P substitutions.
 19. The immunogen of claim 15, wherein the protomers in the recombinant SARS-CoV S ectodomain trimer comprise the amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence at least 90% identical thereto.
 20. The immunogen of claim 19, wherein the protomers in the recombinant SARS-CoV S ectodomain trimer comprise residues 14-1190 of SEQ ID NO: 7, or an amino acid sequence at least 90% identical thereto wherein the protomers comprise the one or two proline substitutions for stabilization in the ectodomain trimer in the prefusion conformation.
 21. The immunogen of claim 15, wherein a C-terminal residue of the protomers in the ectodomain is linked to a trimerization domain by a peptide linker, or is directly linked to the trimerization domain.
 22. The immunogen of claim 21, wherein the trimerization domain is a T4 fibritin trimerization domain.
 23. The immunogen of claim 22, wherein the protomers linked to the T4 fibritin trimerization domain in the recombinant coronavirus S ectodomain trimer comprise residues 14-1217 of SEQ ID NO: 30 (SARS-2P), or an amino acid sequence at least 90% identical to any one of residues 14-1217 of SEQ ID NO: 30 (SARS-2P), wherein the protomers comprise the one or two proline substitutions for stabilization in the ectodomain trimer in the prefusion conformation.
 24. The immunogen of claim 1, wherein the recombinant coronavirus S ectodomain trimer is: a recombinant HKU1-CoV S ectodomain trimer, and the one or two proline substitutions are between residues 1050 to 1070, residues 1060 to 1070, and/or residues 1065 to 1070 of the protomers in the trimer; a recombinant OC43-CoV S ectodomain trimer, and the one or two proline substitutions are between residues 1062 to 1082, residues 1072 to 1082, and/or residues 1077 to 1082 of the protomers in the trimer; a recombinant HKU9-CoV S ectodomain trimer, and the one or two proline substitutions are between residues 966 to 986, residues 976 to 986, and/or residues 981 to 986 of the protomers in the trimer; a recombinant NL63-CoV S ectodomain trimer, and the one or two proline substitutions are between residues 1035 to 1055, residues 1045 to 1055, and/or residues 1050 to 1055 of the protomers in the trimer; a recombinant 229E-CoV S ectodomain trimer, and the one or two proline substitutions are between residues 852 to 872, residues 862 to 872, and/or residues 867 to 872 of the protomers in the trimer; a recombinant WIV1-CoV S ectodomain trimer, and the one or two proline substitutions are between residues 952 to 972, residues 962 to 972, and/or residues 967 to 972 of the protomers in the trimer; a recombinant MHV S ectodomain trimer, and the one or two proline substitutions are between residues 1056 to 1076, residues 1066 to 1076, and/or residues 1071 to 1076 of the protomers in the trimer; a recombinant PEDV S ectodomain trimer, and the one or two proline substitutions are between residues 1059 to 1079, residues 1069 to 1079, and/or residues 1073 to 1079 of the protomers in the trimer; or a recombinant SDCV S ectodomain trimer, and the one or two proline substitutions are between residues 838 to 858, residues 848 to 858, and/or residues 854 to 858 of the protomers in the trimer.
 25. The immunogen of claim 24, wherein the recombinant coronavirus S ectodomain trimer is: a recombinant HKU1-CoV S ectodomain trimer, and the one or two proline substitutions comprise N1067P and/or L1068P substitutions; a recombinant OC43-CoV S ectodomain trimer, and the one or two proline substitutions comprise A1079P and/or L1080P substitutions; a recombinant HKU9-CoV S ectodomain trimer, and the one or two proline substitutions comprise G1018P and/or L1019P substitutions; a recombinant NL63-CoV S ectodomain trimer, and the one or two proline substitutions comprise S1052P and/or I1053P substitutions; a recombinant 229E-CoV S ectodomain trimer, and the one or two proline substitutions comprise I869P and/or I870P substitutions; a recombinant WIV1-CoV S ectodomain trimer, and the one or two proline substitutions comprise K969P and/or V970P substitutions; a recombinant MHV S ectodomain trimer, and the one or two proline substitutions comprise A1073P and/or L1074P substitutions; a recombinant PEDV S ectodomain trimer, and the one or two proline substitutions comprise I1076P and L1077P substitutions; or a recombinant SDCV S ectodomain trimer, and the one or two proline substitutions comprise E855P and V856P substitutions.
 26. The immunogen of claim 24, wherein the protomers in the recombinant coronavirus S ectodomain trimer comprise the amino acid sequence set forth as the ectodomain of any one of: SEQ ID NO: 9 (HKU1-2P), SEQ ID NO: 11 (OC43-2P), SEQ ID NO: 13 (HKU9-2P), SEQ ID NO: 15 (WIV1-2P), SEQ ID NO: 17 (MHV-2P), SEQ ID NO: 19 (NL63-2P), SEQ ID NO: 21 (229E-2P), SEQ ID NO: 39 (PEDV-2P), or SEQ ID NO: 42 (SDCV-2P), or an amino acid sequence at least 90% identical thereto wherein the protomers comprise the one or two proline substitutions for stabilization in the ectodomain trimer in the prefusion conformation.
 27. The immunogen of claim 26, wherein the protomers in the recombinant coronavirus S ectodomain trimer comprise any one of: residues 14-1276 of SEQ ID NO: 9 (HKU1-2P), residues 15-1287 of SEQ ID NO: 11 (OC43-2P), residues 15-1207 of SEQ ID NO: 13 (HKU9-2P), residues 16-1191 of SEQ ID NO: 15 (WIV1-2P), residues 15-1297 of SEQ ID NO: 17 (MHV-2P), residues 16-1291 of SEQ ID NO: 19 (NL63-2P), residues 17-1108 of SEQ ID NO: 21 (229E-2P), residues 21-1322 of SEQ ID NO: 39 (PEDV-2P), or residues 20-1093 of SEQ ID NO: 42 (SDCV-2P); or an amino acid sequence at least 90% identical to any one of residues 14-1276 of SEQ ID NO: 9 (HKU1-2P), residues 15-1287 of SEQ ID NO: 11 (OC43-2P), residues 15-1207 of SEQ ID NO: 13 (HKU9-2P), residues 16-1191 of SEQ ID NO: 15 (WIV1-2P), residues 15-1297 of SEQ ID NO: 17 (MHV-2P), residues 16-1291 of SEQ ID NO: 19 (NL63-2P), residues 17-1108 of SEQ ID NO: 21 (229E-2P), residues 21-1322 of SEQ ID NO: 39 (PEDV-2P), or residues 20-1093 of SEQ ID NO: 42 (SDCV-2P), wherein the protomers comprise the one or two proline substitutions for stabilization in the ectodomain trimer in the prefusion conformation.
 28. The immunogen of claim 24, wherein a C-terminal residue of the protomers in the ectodomain is linked to a trimerization domain by a peptide linker, or is directly linked to the trimerization domain.
 29. The immunogen of claim 28, wherein the trimerization domain is a T4 fibritin trimerization domain.
 30. The immunogen of claim 29, wherein the protomers linked to the T4 fibritin trimerization domain in the recombinant coronavirus S ectodomain trimer comprise any one of residues 14-1303 of SEQ ID NO: 31 (HKU1-2P), residues 15-1234 of SEQ ID NO: 32 (OC43-2P), residues 15-1314 of SEQ ID NO: 33 (HKU9-2P), residues 16-1218 of SEQ ID NO: 34 (WIV1-2P), residues 15-1324 of SEQ ID NO: 35 (MHV-2P), residues 16-1318 of SEQ ID NO: 36 (NL63-2P), residues 17-1135 of SEQ ID NO: 37 (229E-2P), residues 21-1349 of SEQ ID NO: 40, or residues 20-1120 of SEQ ID NO: 43 (SDCV 2P); or an amino acid sequence at least 90% identical to any one of residues 14-1303 of SEQ ID NO: 31 (HKU1-2P), residues 15-1234 of SEQ ID NO: 32 (OC43-2P), residues 15-1314 of SEQ ID NO: 33 (HKU9-2P), residues 16-1218 of SEQ ID NO: 34 (WIV1-2P), residues 15-1324 of SEQ ID NO: 35 (MHV-2P), residues 16-1318 of SEQ ID NO: 36 (NL63-2P), residues 17-1135 of SEQ ID NO: 37 (229E-2P), residues 21-1349 of SEQ ID NO: 40, or residues 20-1120 of SEQ ID NO: 43 (SDCV 2P), wherein the protomers comprise the one or two proline substitutions for stabilization in the ectodomain trimer in the prefusion conformation.
 31. The immunogen of claim 1, wherein the protomers of the recombinant coronavirus S ectodomain trimer further comprise one or more additional amino acid substitutions that stabilize the recombinant coronavirus S ectodomain trimer in the prefusion conformation.
 32. The immunogen of claim 1, wherein a S1/S2 protease cleavage site and/or a S2′ protease cleavage site of the S ectodomain are mutated to inhibit protease cleavage.
 33. The immunogen of claim 1, wherein the recombinant coronavirus S ectodomain trimer is soluble.
 34. The immunogen of claim 1, wherein a C-terminal residue of the protomers in the ectodomain is linked to a transmembrane domain by a peptide linker, or is directly linked to the transmembrane domain.
 35. The immunogen of claim 1, wherein a C-terminal residue of the S2 ectodomain is linked to a protein nanoparticle subunit by a peptide linker, or is directly linked to the protein nanoparticle subunit.
 36. The immunogen of claim 35, wherein the protein nanoparticle subunit is a ferritin nanoparticle subunit.
 37. A protein nanoparticle, comprising the immunogen of claim
 35. 38. A virus-like particle comprising the immunogen of claim
 1. 39. An isolated nucleic acid molecule encoding a protomer of the recombinant coronavirus S ectodomain trimer of claim
 1. 40. The nucleic acid molecule of claim 39, operably linked to a promoter.
 41. The nucleic acid molecule of claim 39, wherein the nucleic acid molecule is an RNA molecule.
 42. A vector comprising the nucleic acid molecule of claim
 39. 43. The vector of claim 42, wherein the vector is a viral vector.
 44. An immunogenic composition comprising the immunogen of claim 1, and a pharmaceutically acceptable carrier.
 45. A method of producing a recombinant coronavirus S ectodomain trimer stabilized in a prefusion conformation, comprising: expressing the nucleic acid molecule or vector of claim 39 in a host cell to produce the recombinant coronavirus S ectodomain trimer; and purifying the recombinant coronavirus S ectodomain trimer.
 46. The recombinant coronavirus S ectodomain trimer produced by the method of claim
 45. 47. A method for generating an immune response to an coronavirus S ectodomain in a subject, comprising administering to the subject an effective amount of the immunogen of claim 1 to generate the immune response.
 48. The method of claim 47, wherein the immune response treats or inhibits infection with the coronavirus.
 49. The method of claim 47, wherein generating the immune response inhibits replication of the coronavirus in the subject.
 50. (canceled) 